POLY-N-ACETYL GLUCOSAMINE (PNAG/dPNAG)-BINDING PEPTIDES AND METHODS OF USE THEREOF

ABSTRACT

The present invention relates to peptides, particularly human monoclonal antibodies, that bind specifically to poly-N-acetyl glucosamine (PNAG), such as  Staphylococcal  PNAG, in acetylated, partially acetylated and/or fully deacetylated form. The invention further provides methods for using these peptides in the diagnosis, prophylaxis and therapy of infections by bacteria that express PNAG such as but not limited to  Staphylococci  and  E. coli.  Some antibodies of the invention enhance opsonophagocytic killing and in vivo protection against bacteria that express PNAG such as but not limited to  Staphylococci  and  E. coli.  Compositions of these peptides, including pharmaceutical compositions, are also provided, as are functionally equivalent variants of such peptides.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/824,510 filed on Jun. 28, 2010, which is a divisionalapplication of U.S. patent application Ser. No. 11/111,688, filed onApr. 21, 2005, now U.S. Pat. No. 7,786,255, which claims priority toU.S. Provisional Application Ser. No. 60/564,105, filed Apr. 21, 2004,the entirety of all above-referenced applications are incorporated byreference herein.

GOVERNMENT SUPPORT

This work was funded in part by grant number AI46706, from the NationalInstitutes of Health. Accordingly, the United States Government may havecertain rights to this invention.

INCORPORATION OF ELECTRONICALLY SUBMITTED SEQUENCE LISTING

The entirety of the Sequence Listing submitted electronically at thesame time of the filing of the instant application is incorporated byreference herein.

FIELD

This invention relates generally to peptides that bind to poly-N-acetylglucosamine (PNAG) and deacetylated PNAG (dPNAG) of bacteria such asStaphylococcus, and their use in the diagnosis and treatment ofStaphylococcal and other PNAG-expressing bacterial infections.

BACKGROUND

Staphylococci are gram-positive bacteria which normally inhabit andcolonize the skin and mucus membranes of humans. If the skin or mucusmembrane becomes damaged during surgery or other trauma, theStaphylococci may gain access to internal tissues causing infection todevelop. If the Staphylococci proliferate locally or enter the lymphaticor blood system, serious infectious complications such as thoseassociated with Staphylococcal bacteremia may result. Complicationsassociated with Staphylococcal bacteremia include septic shock,endocarditis, arthritis, osteomyelitis, pneumonia, and abscesses invarious organs.

Staphylococci include both coagulase positive organisms that produce afree coagulase and coagulase negative organisms that do not produce thisfree coagulase. Staphylococcus aureus is the most commoncoagulase-positive form of Staphylococci. S. aureus generally causesinfection at a local site, either extravascular or intravascular, whichultimately may result in bacteremia. S. aureus is also a leading causeof acute osteomyelitis and causes Staphylococcal pneumonia infections.Additionally, S. aureus is responsible for approximately 1-9% of thecases of bacterial meningitis and 10-15% of brain abscesses.

There are at least twenty-one known species of coagulase-negativeStaphylococci, including S. epidermidis, S. saprophyticus, S. hominis,S. warneri, S. haemolyticus, S. saprophiticus, S. cohnii, S. xylosus, S.simulans, and S. capitis. S. epidermidis is the most frequentinfection-causing agent associated with intravenous access devices andthe most frequent isolate in primary nosocomial bacteremias. S.epidermidis is also associated with prosthetic valve endocarditis.

Staphylococcus is also a common source of bacterial infections inanimals. For instance, Staphylococcal mastitis is a common problem inruminants including cattle, sheep, and goats. The disease is generallytreated with antibiotics to reduce the infection but the treatment is acostly procedure and still results in a loss of milk production. Themost effective vaccines for livestock identified to date are live,intact S. aureus vaccines administered subcutaneously. Theadministration of live vaccines, however, is associated with the risk ofinfection and with toxic reactions. For that reason, many researchershave attempted to produce killed S. aureus vaccines and/or to isolatecapsular polysaccharides or cell wall components which will induceimmunity to S. aureus. None of these attempts, however, has beensuccessful.

SUMMARY

The present invention relates generally to the identification and use ofpeptides that bind to poly-N-acetyl glucosamine (PNAG) such asStaphylococcal poly-N-acetyl glucosamine (PNAG), and poorly acetylatedor deacetylated PNAG (collectively referred to herein as dPNAG). Thesepeptides are referred to herein as PNAG/dPNAG-binding peptides. Examplesof such peptides include those having amino acid sequences derived fromcomplementarity determining regions (CDRs) or variable regions ofantibodies described herein or produced from hybridomas deposited withthe American Type Culture Collection (ATCC), located at 10801 UniversityBlvd. Manassas, Va. 20110-2209 on Apr. 21, 2004, under Accession Nos.PTA-5931 (F598), PTA-5932 (F628) and PTA-5933 (F630). These peptidesinclude but are not limited to polypeptides, monoclonal antibodies (suchas human monoclonal antibodies) and antibody fragments. A common featureof the peptides disclosed herein is their ability to recognize and bindto Staphylococcal PNAG and/or dPNAG specifically. PNAG and/or dPNAGexpressed by other bacterial strains may also be recognized and bound bythe peptides of the invention. An important characteristic of some ofthe antibodies and antibody fragments provided by the invention is theirability to enhance opsonization and phagocytosis (i.e.,opsonophagocytosis) of bacterial strains, such as Staphylococcalspecies, that express PNAG.

Thus, in one aspect, the invention provides a composition comprising anisolated peptide that selectively binds to Staphylococcal poly-N-acetylglucosamine (PNAG/dPNAG) and comprises an amino acid sequence of aStaphylococcal PNAG/dPNAG-binding CDR, or functionally equivalentvariant thereof.

Various embodiments are shared between this and other aspects of theinvention. These embodiments will be recited once but it is to beunderstood that they apply equally to all aspects of the invention.

In one embodiment, the Staphylococcal PNAG/dPNAG-binding CDR is aStaphylococcal PNAG/dPNAG-binding CDR3. The StaphylococcalPNAG/dPNAG-binding CDR3 may comprise an amino acid sequence of a heavychain CDR3 selected from the group consisting of SEQ ID NO: 9, SEQ IDNO:15 and SEQ ID NO:21 or it may comprise an amino acid sequence of aheavy chain CDR3 derived from a deposited hybridoma having ATCCAccession No. PTA-5931, PTA-5932 or PTA-5933. The StaphylococcalPNAG/dPNAG-binding CDR3 may comprise an amino acid sequence of a lightchain CDR3 selected from the group consisting of SEQ ID NO:12, SEQ IDNO:18, and SEQ ID NO: 24 or it may comprise an amino acid sequence of alight chain CDR3 derived from a deposited hybridoma having ATCCAccession No. PTA-5931, PTA-5932 or PTA-5933.

In another embodiment, the Staphylococcal PNAG/dPNAG-binding CDR is aStaphylococcal PNAG/dPNAG-binding CDR2. The StaphylococcalPNAG/dPNAG-binding CDR2 may comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14,SEQ ID NO:17, SEQ ID NO:20 and SEQ ID NO:23 or it may comprise an aminoacid sequence of a CDR2 derived from a deposited hybridoma having ATCCAccession No. PTA-5931, PTA-5932 or PTA-5933.

In another embodiment, the Staphylococcal PNAG/dPNAG-binding CDR is aStaphylococcal PNAG/dPNAG-binding CDR1. The StaphylococcalPNAG/dPNAG-binding CDR1 may comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:16, SEQ ID NO:19 and SEQ ID NO:22 or it may comprise an aminoacid sequence of a CDR1 derived from a deposited hybridoma having ATCCAccession No. PTA-5931, PTA-5932 or PTA-5933.

In one embodiment, the isolated peptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQID NO:5 or an amino acid sequence of a heavy chain variable regionderived from a deposited hybridoma having ATCC Accession No. PTA-5931,PTA-5932 or PTA-5933.

In another embodiment, the isolated peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4and SEQ ID NO:6 or an amino acid sequence of a light chain variableregion derived from a deposited hybridoma having ATCC Accession No.PTA-5931, PTA-5932 or PTA-5933.

In one embodiment, the isolated peptide is an isolated antibody orantibody fragment, such as but not limited to an isolated intact,preferably soluble, monoclonal antibody or an isolated monoclonalantibody fragment such as but not limited to an F(ab′)₂ fragment an Fdfragment and an Fab fragment. The isolated antibody may be an antibodyproduced from a deposited hybridoma having ATCC Accession No. PTA-5931,PTA-5932 or PTA-5933, or an antibody fragment thereof.

In one embodiment, the isolated antibody or antibody fragment enhancesopsonophagocytosis of PNAG-expressing bacterial strains (e.g.,Staphylococci such as but not limited to S. aureus or S. epidermidis).

In one embodiment, the isolated antibody or antibody fragment comprisesan amino acid sequence comprising a heavy chain variable region andselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQID NO:5, and an amino acid sequence comprising a light chain variableregion and selected from the group consisting of SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:6. In another embodiment, the isolated antibody orantibody fragment comprises an amino acid sequence comprising a heavychain variable region derived from a deposited hybridoma having ATCCAccession No. PTA-5931, PTA-5932 or PTA-5933, and an amino acid sequencecomprising light chain variable region derived from a depositedhybridoma having ATCC Accession No. PTA-5931, PTA-5932 or PTA-5933.

The isolated antibody or antibody fragment may comprise an amino acidsequence of SEQ ID NO:1 and an amino acid sequence of SEQ ID NO:2, or anamino acid sequence of SEQ ID NO:3 and an amino acid sequence of SEQ IDNO:4, or an amino acid sequence of SEQ ID NO:5 and an amino acidsequence of SEQ ID NO:6.

The isolated antibody or antibody fragment may comprise an amino acidsequence of a heavy chain variable region derived from depositedhybridoma having Accession No. PTA-5931 (F598) and an amino acidsequence comprising light chain variable region derived from depositedhybridoma having Accession No. PTA-5931 (F598), or an amino acidsequence of a heavy chain variable region derived from depositedhybridoma having Accession No. PTA-5932 (F628) and an amino acidsequence comprising light chain variable region derived from depositedhybridoma having Accession No. PTA-5932 (F628), or an amino acidsequence of a heavy chain variable region derived from depositedhybridoma having Accession No. PTA-5933 (F630) and an amino acidsequence comprising light chain variable region derived from depositedhybridoma having Accession No. PTA-5933 (F630).

In one embodiment, the isolated peptide is conjugated to a detectablelabel. The detectable label may be an in vivo or an in vitro detectablelabel.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier. In other embodiments, the isolated peptide such asthe isolated antibody or antibody fragment is present in an effectiveamount for inhibiting an infection by a bacterial strain expressing PNAG(such as a Staphylococcal infection) or in an effective amount fordetecting a bacterial strain expressing PNAG (such as Staphylococci) ina sample in or from a subject.

In one embodiment, the isolated peptide selectively binds toStaphylococcal PNAG. In another embodiment, the isolated peptideselectively binds to Staphylococcal dPNAG.

In yet another aspect, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a StaphylococcalPNAG/dPNAG-binding CDR.

In one embodiment, the nucleotide sequence is selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO: 18,SEQ ID NO:21 and SEQ ID NO:24. In another embodiment, the nucleotidesequence is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

In one embodiment, the nucleic acid is a heavy chain variable regionnucleic acid molecule derived from a hybridoma having Accession No.PTA-5931, PTA-5932 or PTA-5933. In another embodiment, the nucleic acidis a light chain variable region nucleic acid molecule derived from ahybridoma having Accession No. PTA-5931, PTA-5932 or PTA-5933. In yetanother embodiment, the nucleic acid is a heavy chain CDR nucleic acidmolecule derived from a hybridoma having Accession No. PTA-5931,PTA-5932 or PTA-5933 or it is a light chain CDR nucleic acid moleculederived from a hybridoma having Accession No. PTA-5931, PTA-5932 orPTA-5933.

The invention further provides, in other aspects, expression vectorscomprising the afore-mentioned isolated nucleic acid molecules, operablylinked to a promoter and cells transformed or transfected with suchexpression vectors.

In other aspects, the invention provides an isolated cell producing ananti-Staphylococcal PNAG/dPNAG monoclonal antibody (F598) and havingATCC Accession No. PTA-5931, an isolated cell producing ananti-Staphylococcal PNAG/dPNAG monoclonal antibody (F628) and havingATCC Accession No. PTA-5932, and an isolated cell producing ananti-Staphylococcal PNAG/dPNAG monoclonal antibody (F630) and havingATCC Accession No. PTA-5933. The invention further provides, inadditional aspects, the isolated monoclonal antibody produced by theafore-mentioned deposited isolated cells, or antibody fragments thereof.The antibody fragment may be but it not limited to an F(ab′)₂ fragment,an Fd fragment or an Fab fragment. In a related embodiment, the fragmentenhances opsonophagocytosis of PNAG-expressing bacterial strains (e.g.,Staphylococci such as but not limited to S. aureus or S. epidermidis).

In another aspect, the invention provides a method for detectingbacterial strains expressing PNAG (such as Staphylococci) in a subjector a sample from a subject. The method comprises determining a testlevel of binding of an isolated peptide or a functionally equivalentvariant thereof to a sample in or from a subject, and comparing the testlevel of binding to a control, wherein the isolated peptide selectivelybinds to Staphylococcal PNAG/dPNAG and comprises a StaphylococcalPNAG/dPNAG-binding CDR, or a functionally equivalent variant thereof,and wherein a test level of binding that is greater than the control isindicative of the presence of the bacterial strain (e.g., Staphylococci)in the sample. The bacteria to be detected may be Staphylococci, E.coli, Yersinia pestis (Y. pestis), Y. entercolitica, Xanthomonasaxonopodis (X. axonopodis), Pseudomonas fluorescens (P. fluorescens),Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), A.pleuropneumoniae, Bordetella pertussis (B. pertussis), B. parapertussisor B. bronchiseptica. The invention also provides methods for detectingand treating plant infections by bacteria expressing PNAG such asRalstonia solanacearum (R. solanacearum).

In one embodiment, the test level of binding is measured in vitro.

In another aspect, the invention provides a method for treating asubject having, or at risk of developing, an infection by a bacterialstrain expressing PNAG (e.g., a Staphylococcal infection). The methodcomprises administering to a subject in need of such treatment anisolated peptide that selectively binds to Staphylococcal PNAG/dPNAG,and comprises a Staphylococcal PNAG/dPNAG-binding CDR or a functionallyequivalent variant thereof, in an amount effective to inhibit theinfection. In another embodiment, the isolated peptide is conjugated toa cytotoxic agent.

In one embodiment, the subject has or is at risk of developing aStaphylococcal infection, such as but not limited to S. aureus or S.epidermidis infection. In another embodiment, the subject has or is atrisk of developing an E. coli, Yersinia pestis (Y. pestis), Y.entercolitica, Xanthomonas axonopodis (X. axonopodis), Pseudomonasfluorescens (P. fluorescens), Actinobacillus actinomycetemcomitans (A.actinomycetemcomitans), A. pleuropneumoniae, Bordetella pertussis (B.pertussis), B. parapertussis or B. bronchiseptica infection.

The foregoing bacterial infections underlie conditions such asgastroenteritis, urinary-tract infections, plague, whopping cough,bloodstream infections and dental infections (periodontitis). Theinvention intends to treat these latter conditions by treatingunderlying the bacterial infection. The detection and treatment methodsprovided herein are suitable for human and non-human subjects that haveor are at risk of developing such infections. Non-human subjects includeagricultural animals such as cows and pigs, but are not so limited.

Ralstonia solanacearum (R. solanacearum) is another PNAG expressingbacteria, however it is considered a plant rather than an animalpathogen. The invention contemplates detection and treatment of plantspecies having such infections using the binding peptides providedherein, preferably conjugated to a detectable or cytotoxic label,depending on the method.

In yet another aspect, the invention provides a method for treating aninfection by a bacterial strain that expresses PNAG (e.g.,Staphylococcal infection) comprising administering to a subject in needthereof a PNAG/dPNAG-binding peptide that reduces bacterial load in asubject by at least 50% in at least 4 hours after exposure to abacterium that expresses PNAG in an amount effective to treat theinfection.

In one embodiment, the PNAG/dPNAG-binding peptide is an isolatedantibody or antibody fragment. In one embodiment, the infection is aStaphylococcal infection. In one embodiment, the Staphylococcalinfection is an S. aureus infection or an S. epidermidis infection. Inanother embodiment, the infection is an E. coli, Yersinia pestis (Y.pestis), Y. entercolitica, Xanthomonas axonopodis (X. axonopodis),Pseudomonas fluorescens (P. fluorescens), Actinobacillusactinomycetemcomitans (A. actinomycetemcomitans), A. pleuropneumoniae,Bordetella pertussis (B. pertussis), B. parapertussis or B.bronchiseptica infection. Ralstonia solanacearum (R. solanacearum)infections are also contemplated by the invention, although these affectplants rather than animals. In another embodiment, thePNAG/dPNAG-binding peptide is administered prior to exposure to thebacterium, such as but not limited to at least 24 hours prior toexposure to the bacterium.

In one embodiment, the PNAG/dPNAG-binding peptide reduces bacterial loadin a subject by at least 60% in at least 4 hours after exposure to thebacterium. In another embodiment, the PNAG/dPNAG-binding peptide reducesbacterial load in a subject by at least 50% in 2 hours after exposure tothe bacterium. In yet another embodiment, the PNAG/dPNAG-binding peptidereduces bacterial load in a subject by at least 60% in 2 hours afterexposure to the bacterium. Bacteria that express PNAG include but arenot limited to Staphylococci, E. coli, Yersinia pestis (Y. pestis), Y.entercolitica, Xanthomonas axonopodis (X. axonopodis), Pseudomonasfluorescens (P. fluorescens), Actinobacillus actinomycetemcomitans (A.actinomycetemcomitans), A. pleuropneumoniae, Bordetella pertussis (B.pertussis), B. parapertussis and B. bronchiseptica, which affectanimals, and Ralstonia solanacearum (R. solanacearum) which affectsplants.

These and other embodiments of the invention will be described ingreater detail herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the binding affinities of monoclonalantibodies (MAbs) F598, F628 and F630 (in an IgG2 form) to native PNAG.MAb to P. aeruginosa MEP is used as a negative control.

FIG. 2 is a graph showing the binding affinities of MAbs F598, F628 andF630 (in an IgG2 form) to dPNAG.

FIG. 3 is a graph showing the results of a competition ELISA using PNAGand MAbs F598, F628 and F630 (in an IgG2 form).

FIG. 4 is a graph showing the binding affinities of MAbs F598, F628 andF630 (in an IgG1 form) to native PNAG.

FIG. 5 is a graph showing the binding affinities of MAbs F598, F628 andF630 (in an IgG1 form) to dPNAG.

FIG. 6 is a graph showing complement fixation activity of MAbs F598,F628 and F630 in both IgG1 and IgG2 form on PNAG. MAb to P. aeruginosaMEP is used as a negative control.

FIG. 7 is a graph showing the opsonophagocytic activity of MAbs F598,F628 and F630 in IgG1 and IgG2 form against S. aureus strain Mn8.

FIG. 8A is a bar graph showing averaged results comparing levels ofStaphylococci in the blood of mice (8 per group) given either a controlhuman IgG1 MAb to P. aeruginosa alginate or MAb F598 specific toPNAG/dPNAG (in an IgG1 form) and demonstrating that MAb F598 can providepassive protection against S. aureus challenge.

FIG. 8B is a graph showing the results of protection against S. aureuschallenge in individual mice, reporting the CFU per ml of bloodfollowing administration of a control human IgG1 MAb to P. aeruginosaalginate and MAb F598 specific for PNAG/dPNAG (in an IgG1 form).

FIG. 8C is a graph showing the results of protection against S. aureuschallenge in individual FVB mice using MAb F598 and control MAb to P.aeruginosa MEP.

FIG. 9 is an immunoblot showing PNAG expression by E. coli UTI strainslabeled D-U and including an E. coli pga over-expressing isolate (topright hand corner).

FIG. 10 is a bar graph showing the level of killing of E. coli isolatesusing polyclonal antiserum raised against S. aureus dPNAG.

FIGS. 11A and 11B are graphs showing the level of killing of E. coliisolates expressing relatively high (strain U) and intermediate (strainP) levels of PNAG, respectively, using polyclonal antiserum raisedagainst dPNAG and PNAG.

FIG. 12 is a bar graph showing reduction in CFU from differentPNAG-expressing bacterial strains using F598, F628 and F630.

FIG. 13 is a graph showing proportion of S. aureus bacteria killed byF598 and F628 as a function of icaB gene presence or absence.

FIG. 14 is a graph showing proportion of S. aureus bacteria killed byF598 and F628 as a function of icaB gene over-expression.

It is to be understood that the Figures are not required for enablementof the invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is the amino acid sequence of antibody F598 heavy chainvariable region.

SEQ ID NO: 2 is the amino acid sequence of antibody F598 light chainvariable region.

SEQ ID NO: 3 is the amino acid sequence of antibody F628 heavy chainvariable region.

SEQ ID NO: 4 is the amino acid sequence of antibody F628 light chainvariable region.

SEQ ID NO: 5 is the amino acid sequence of antibody F630 heavy chainvariable region.

SEQ ID NO: 6 is the amino acid sequence of antibody F630 light chainvariable region.

SEQ ID NO: 7 is the amino acid sequence of CDR1 of antibody F598 heavychain.

SEQ ID NO: 8 is the amino acid sequence of CDR2 of antibody F598 heavychain.

SEQ ID NO: 9 is the amino acid sequence of CDR3 of antibody F598 heavychain.

SEQ ID NO: 10 is the amino acid sequence of CDR1 of antibody F598 lightchain.

SEQ ID NO: 11 is the amino acid sequence of CDR2 of antibody F598 lightchain.

SEQ ID NO: 12 is the amino acid sequence of CDR3 of antibody F598 lightchain.

SEQ ID NO: 13 is the amino acid sequence of CDR1 of antibody F628 heavychain.

SEQ ID NO: 14 is the amino acid sequence of CDR2 of antibody F628 heavychain.

SEQ ID NO: 15 is the amino acid sequence of CDR3 of antibody F628 heavychain.

SEQ ID NO: 16 is the amino acid sequence of CDR1 of antibody F628 lightchain.

SEQ ID NO: 17 is the amino acid sequence of CDR2 of antibody F628 lightchain.

SEQ ID NO: 18 is the amino acid sequence of CDR3 of antibody F628 lightchain.

SEQ ID NO: 19 is the amino acid sequence of CDR1 of antibody F630 heavychain.

SEQ ID NO: 20 is the amino acid sequence of CDR2 of antibody F630 heavychain.

SEQ ID NO: 21 is the amino acid sequence of CDR3 of antibody F630 heavychain.

SEQ ID NO: 22 is the amino acid sequence of CDR1 of antibody F630 lightchain.

SEQ ID NO: 23 is the amino acid sequence of CDR2 of antibody F630 lightchain.

SEQ ID NO: 24 is the amino acid sequence of CDR3 of antibody F630 lightchain.

SEQ ID NO: 25 is the nucleotide sequence of antibody F598 heavy chainvariable region.

SEQ ID NO: 26 is the nucleotide sequence of antibody F598 light chainvariable region.

SEQ ID NO: 27 is the nucleotide sequence of antibody F628 heavy chainvariable region.

SEQ ID NO: 28 is the nucleotide sequence of antibody F628 light chainvariable region.

SEQ ID NO: 29 is the nucleotide sequence of antibody F630 heavy chainvariable region.

SEQ ID NO: 30 is the nucleotide sequence of antibody F630 light chainvariable region.

SEQ ID NO: 31 is the nucleotide sequence of CDR1 of antibody F598 heavychain.

SEQ ID NO: 32 is the nucleotide sequence of CDR2 of antibody F598 heavychain.

SEQ ID NO: 33 is the nucleotide sequence of CDR3 of antibody F598 heavychain.

SEQ ID NO: 34 is the nucleotide sequence of CDR1 of antibody F598 lightchain.

SEQ ID NO: 35 is the nucleotide sequence of CDR2 of antibody F598 lightchain.

SEQ ID NO: 36 is the nucleotide sequence of CDR3 of antibody F598 lightchain.

SEQ ID NO: 37 is the nucleotide sequence of CDR1 of antibody F628 heavychain.

SEQ ID NO: 38 is the nucleotide sequence of CDR2 of antibody F628 heavychain.

SEQ ID NO: 39 is the nucleotide sequence of CDR3 of antibody F628 heavychain.

SEQ ID NO: 40 is the nucleotide sequence of CDR1 of antibody F628 lightchain.

SEQ ID NO: 41 is the nucleotide sequence of CDR2 of antibody F628 lightchain.

SEQ ID NO: 42 is the nucleotide sequence of CDR3 of antibody F628 lightchain.

SEQ ID NO: 43 is the nucleotide sequence of CDR1 of antibody F630 heavychain.

SEQ ID NO: 44 is the nucleotide sequence of CDR2 of antibody F630 heavychain.

SEQ ID NO: 45 is the nucleotide sequence of CDR3 of antibody F630 heavychain.

SEQ ID NO: 46 is the nucleotide sequence of CDR1 of antibody F630 lightchain.

SEQ ID NO: 47 is the nucleotide sequence of CDR2 of antibody F630 lightchain.

SEQ ID NO: 48 is the nucleotide sequence of CDR3 of antibody F630 lightchain.

SEQ ID NO: 49 is the nucleotide sequence of primer lambda constant.

SEQ ID NO: 50 is the nucleotide sequence of primer Hu lambda sig 5.

SEQ ID NO: 51 is the nucleotide sequence of primer Heavy chain constant.

SEQ ID NO: 52 is the nucleotide sequence of primer VH7LDRHU.

SEQ ID NO: 53 is the nucleotide sequence of primer Hu lambda sig 1.

SEQ ID NO: 54 is the nucleotide sequence of primer VH1LDRHU.

SEQ ID NO: 55 is the amino acid sequence of F598 heavy chain variableregion including some constant region sequence.

SEQ ID NO: 56 is the nucleotide sequence of F598 heavy chain variableregion including some constant region sequence.

SEQ ID NO: 57 is the amino acid sequence of F598 light chain variableregion including some constant region sequence.

SEQ ID NO: 58 is the amino acid sequence of F628 heavy chain variableregion including some constant region sequence.

SEQ ID NO: 59 is the nucleotide sequence of F628 heavy chain variableregion including some constant region sequence.

SEQ ID NO: 60 is the amino acid sequence of F630 light chain variableregion including some constant region sequence.

SEQ ID NO: 61 is the nucleotide sequence of F630 light chain variableregion including some constant region sequence.

DETAILED DESCRIPTION

The invention provides compositions and methods useful, inter alia, forimmunization of humans and animals against infection by bacterialstrains that express poly-N-acetyl glucosamine (PNAG) as well asdetection of such pathogens. Such bacterial strains include but are notlimited to coagulase-negative and coagulase-positive Staphylococci suchas S. aureus and S. epidermis. The invention further provides peptidesthat bind to various forms of PNAG expressed by some bacterial strains.

The invention is based in part on the discovery, isolation andcharacterization of a number of human monoclonal antibodies that bind tovarious forms of PNAG (including highly acetylated forms, poorlyacetylated forms and deacetylated forms, as described below). Theseantibodies are produced by hybridomas deposited with the ATCC under ATCCAccession Nos. PTA-5931, PTA-5932 and PTA-5933 on Apr. 21, 2004 inaccordance with the Budapest Patent Treaty. The hybridomas and theantibodies they produce are designated F598, F628 and F630. Thesehybridomas are referred to herein repeatedly. It is to be understoodthat reference to hybridomas (or antibodies produced by hybridomas)having ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933 means theafore-mentioned hybridomas. The deposited hybridomas were produced fromB cells harvested from a human subject recovering from a Staphylococcalinfection. The B cells were transformed with the Epstein-Barr virus andthen fused with the human-mouse myeloma cell line HMMA 2.5 to generatethe deposited hybridomas.

PNAG exists in nature in various forms that differ according to thedegree of acetate substitutions. Acetate substitutions can range from0-100%. As used herein, PNAG refers to “native PNAG” corresponding tothe naturally occurring mixture of PNAG with the aforementioned range ofacetate substitutions. Poorly acetylated PNAG is a subpopulation of PNAGpolysaccharides in which less than 50% of amino groups of glucosamineare substituted with acetate. As used herein, the term dPNAG embracesboth poorly acetylated PNAG as well as completely deacetylated PNAG(i.e., dPNAG refers to a subset of PNAG polysaccharides that comprise0-less than 50% acetate substituents).

PNAG has the following structure:

where, n is an integer ranging from 2 to greater than or equal to 300, Ris selected from the group consisting of —NH—CO—CH₃ and —NH₂. PNAG has abeta (β) 1-6 linkage (i.e., it is comprised of glucosamine monomer unitslinked together by beta (β) 1-6 linkages).

PNAG may be a homo-polymer. A homo-polymer is one in which the R groupsof the glucosamine residues are identical. The homo-polymer may comprisesolely unsubstituted R groups (i.e., R═NH₂). PNAG can also be ahetero-polymer with a mixture of —NH₂ and —NH—CO—CH₃ groups at the Rposition. dPNAG has the identical structure as PNAG with the exceptionthat less than 50% of the R groups are —NH—CO—CH₃.

PNAG and dPNAG can be naturally occurring and prepared from anybacterial strain carrying the ica locus (or a homologous locus such asthe pga locus), producing the biosynthetic enzymes encoded by thislocus, and using these enzymes to synthesize PNAG or dPNAG. Bacteriathat express PNAG include Staphylococci such as S. aureus and S.epidermidis, E. coli such as E. coli strains O157:H7 and CFT073,Yersinia pestis, Yersinia entercolitica, Xanthomonas axonopodis,Pseudomonas fluorescens (all of which are sequenced species withcomplete pgaABCD loci), and Actinobacillus actinomycetemcomitans (AA),Actinobacillus pleuropneumoniae (Ap), Ralstonia solanacearum (e.g.,megaplasmid form), Bordetella pertussis, Bordetella parapertussis andBordetella bronchiseptica (all of which contain pgaABC genes butapparently lack a pgaD homologue). pgaD apparently is not required forPNAG expression as pgaABC encoding species such as AA and Ap (listedabove) make PNAG.

Bacteria that express PNAG are bacteria that carry the ica locus or ahomologous locus such as the pga locus. For example, PNAG-expressingStaphylococci are Staphylococci that carry the ica locus.PNAG-expressing bacterial strains include dPNAG-expressing bacterialstrains. For example, PNAG-expressing Staphylococci includedPNAG-expressing Staphylococci. These strains include but are notlimited to S. epidermis and S. aureus, as well as other strains (e.g.,S. carnosus) that have been transformed with the genes in the ica locusor homologous locus such as the pga locus. In particular, PNAG can beprepared from specific strains including S. epidermis RP62A (ATCC number35984), S. epidermis RP12 (ATCC number 35983), S. epidermis M187, S.carnosus TM300 (pCN27), S. aureus RN4220 (pCN27), S. aureus MN8 mucoid,E. coli O157:H7 and E. coli CFT073. dPNAG may also be synthesized denovo or via modification of native PNAG. PNAG and dPNAG can be preparedaccording to the methods described in Maira-Litran et al. Infect Immun.2002 August; 70(8):4433, and in U.S. patent application Ser. No.10/713,790 filed on Nov. 12, 2003.

PNAG is also expressed by other bacteria including but not limited to E.coli, Yersinia pestis (Y. pestis), Y. entercolitica, Xanthomonasaxonopodis (X. axonopodis), Pseudomonas fluorescens (P. fluorescens),Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), A.pleuropneumoniae, Ralstonia solanacearum (R. solanacearum), Bordetellapertussis (B. pertussis), B. parapertussis and B. bronchiseptica. Asdescribed in the Examples, 17 out of 18 urinary tract infection E. coliisolates carried the pga locus. Of these, about one third expressedrelatively high levels of PNAG, about one third expressed relativelyintermediate levels of PNAG, and the remaining third expressedrelatively low levels of PNAG. The above analyses were carried out byimmunoblot using antisera raised to S. aureus PNAG. This is evidencethat PNAG from one species can be used to raise antibodies (andaccordingly binding peptides) to other species that express PNAG.

Thus, in one aspect, the invention provides binding peptides andantibodies. The antibodies of the invention bind to StaphylococcalPNAG/dPNAG and enhance opsonophagocytosis of species that elaborate PNAG(i.e., opsonophagocytic human monoclonal antibodies specific forStaphylococcal PNAG/dPNAG). The antibodies are referred to herein asanti-Staphylococcal PNAG/dPNAG antibodies. It is to be understood,however, that such antibodies are able to bind PNAG/dPNAG regardless ofits source. Accordingly, antibodies of the invention that are defined asbinding to, for example, Staphylococcal PNAG/dPNAG and capable ofdetecting and/or enhancing opsonophagocytosis of, for example,Staphylococcal species are also capable of detecting and/or enhancingopsonophagocytosis of non-Staphylococcal PNAG-expressing bacteria.

An anti-Staphylococcal PNAG/dPNAG antibody is an antibody that a) bindsto both PNAG and dPNAG, b) binds to PNAG but not dPNAG, or c) binds todPNAG but not PNAG. Preferred antibodies bind to dPNAG.

Antibodies F598, F628 and F630 are all able to bind to native PNAG andsome are also able to bind to dPNAG. Although not intending to be boundby any mechanism or theory, it is believed that antibodies thatrecognize dPNAG are more likely to bind specifically to parts of thePNAG molecule that do not contain acetate groups, rather than to partsof the molecule that include substituents such as the acetatesubstitutions. For example, antibodies that bind to dPNAG may recognizeand bind to the backbone of PNAG rather than its acetate substituents.These antibodies are capable of mediating opsonophagocytic killing ofPNAG-expressing bacteria such as but not limited to Staphylococcal or E.coli isolates from infected human subjects. When used in vivo in murinemodels of Staphylococcal infection, the antibodies provide protection toStaphylococcal challenge. The conditions under which each monoclonalantibody provides protection may vary. These and other findings aredescribed in greater detail in the Examples.

Although not intending to be bound by any particular theory, it isbelieved that progression of infection by PNAG-expressing bacteria (suchas Staphylococcal infection) is due to a failure to produce an adequateimmune response that eliminates the pathogen. Specifically, one of thedefects is a failure to produce opsonophagocytic antibodies specific forPNAG (such as that produced by Staphylococci.)

Opsonophagocytic antibodies are antibodies that deposit themselves ontoan antigen or onto a bacterium with and without the ability to recruitadditional deposition of components of the complement system andfacilitate the phagocytosis of the antigen or bacterium by phagocyticcells such as antigen presenting cells (e.g., macrophages or dendriticcells), or polymorphonuclear neutrophils. Phagocytosis can proceed in anFc-mediated manner that involves only the antibody bound to the antigenor bacterium. Phagocytosis can also proceed by binding of complementreceptors on phagocytes to complement opsonins on bacterial surfaces towhich antibodies have deposited. Phagocytosis can also proceed by acombination of these two mechanisms. The ability to provideopsonophagocytic antibodies to the site of infection should thereforecontribute to the eradication of the infection more effectively thanpreviously possible.

Both PNAG and dPNAG are highly immunogenic in vivo and are capable ofeliciting antibodies that mediate opsonic killing and protection frominfection, it is hypothesized that dPNAG preferentially elicitsantibodies that mediate opsonic killing and protection from infection.The dPNAG polysaccharide is therefore useful, inter alia, in thegeneration of immune responses, including antibody dependent immuneresponses, to PNAG-expressing bacterial strains such as but not limitedto Staphylococci. The antibodies elicited following dPNAG administrationrecognize dPNAG and in important embodiments also recognize highlyacetylated forms of PNAG.

Thus, the invention relates to the identification and use of peptidesthat bind to PNAG and/or dPNAG. Peptides that bind to StaphylococcalPNAG and/or dPNAG are referred to herein as PNAG/dPNAG-binding peptides.Again, it is to be understood that such binding peptides are able tobind PNAG/dPNAG regardless of source. PNAG/dPNAG-binding peptidesinclude a) peptides that bind to both PNAG and dPNAG, b) peptides thatbind to PNAG and not to dPNAG (referred to herein as PNAG-bindingpeptides), and c) peptides that bind to dPNAG and not to PNAG (referredto herein as dPNAG-binding peptides). In preferred embodiments, thepeptides at least bind to dPNAG (thereby embracing afore-mentionedcategories (a) and (c)).

The peptides of the invention minimally comprise regions that bind toPNAG/dPNAG (i.e., Staphylococcal PNAG/dPNAG-binding regions). As usedherein, a Staphylococcal PNAG/dPNAG-binding region is a region that a)binds to both PNAG and dPNAG, b) binds to PNAG but not dPNAG (referredto herein as a PNAG-binding region), or c) binds to dPNAG but not PNAG(referred to herein as a dPNAG-binding region), regardless of the sourceof PNAG/dPNAG. Preferably, the PNAG/dPNAG binding region is a regionthat at least binds dPNAG (and therefore embraces categories (a) and(c)). Staphylococcal PNAG/dPNAG-binding regions derive from thePNAG/dPNAG-binding regions of the antibodies of the invention, oralternatively, they are functionally equivalent variants of suchregions.

Accordingly, two particularly important classes of antibody-derivedPNAG/dPNAG-binding regions are variable regions and CDRs of theantibodies described herein or produced by hybridomas deposited with theATCC under ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933 on Apr.21, 2004. CDR and variable region nucleic acids can be cloned fromantibody-producing cells such as those on deposit as described in theExamples.

An antibody, as is well known in the art, is an assembly of polypeptidechains linked by disulfide bridges. Two principle amino acid chains,referred to as the light chain and heavy chain, make up all majorstructural isotypes of antibody. Both heavy chains and light chains arefurther divided into subregions referred to as variable regions andconstant regions. In some instances, the peptides encompass the antibodyheavy and light chain variable regions of the foregoing antibodies. Theheavy chain variable region is a peptide which generally ranges from 100to 150 amino acids in length. The light chain variable region is apeptide which generally ranges from 80 to 130 amino acids in length.

As is also well-known in the art, CDRs of an antibody are the portionsof the antibody variable region which are largely responsible for thebinding specificity of an antibody for a given antigen or antigenicepitope. The CDRs directly interact with the epitope of the antigen(see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain andthe light chain variable regions of IgG immunoglobulins, there are fourframework regions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1, CDR 2 and CDR3). Theframework regions (FRs) maintain the tertiary structure of the paratope,which is the portion of the antibody which is involved in theinteraction with the antigen or antigenic epitope. CDRs, and inparticular CDR3, and more particularly heavy chain CDR3, contributesubstantially to antibody specificity. Because CDRs, and in particularCDR3, confer a large proportion of antigenic specificity on theantibody, these regions may be incorporated into other antibodies orpeptides to confer the identical antigenic specificity onto thatantibody or peptide.

Preferably, the PNAG/dPNAG-binding peptides minimally encompass at leastone CDR from those described herein or those that can be derived fromthe deposited hybridomas (i.e., a Staphylococcal PNAG/dPNAG-bindingCDR). As used herein, a Staphylococcal PNAG/dPNAG-binding CDR is a CDRdescribed herein or is a CDR derived from hybridomas deposited underATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933. StaphylococcalPNAG/dPNAG-binding CDRs include a) CDRs that bind to both PNAG anddPNAG, b) CDRs that bind to PNAG and not to dPNAG (referred to herein asPNAG-binding CDRs), and c) CDRs that bind to dPNAG and not to PNAG(referred to herein as dPNAG-binding CDRs), regardless of the source ofthe PNAG/dPNAG. These peptides preferably contain at least oneStaphylococcal PNAG/dPNAG-binding CDR.

The Staphylococcal PNAG/dPNAG-binding region may be a StaphylococcalPNAG/dPNAG-binding CDR1, a Staphylococcal PNAG/dPNAG-binding CDR2, or aStaphylococcal PNAG/dPNAG-binding CDR3, all of which are derived fromthe antibodies and antibody variable chains disclosed herein.

As used herein, a “Staphylococcal PNAG/dPNAG-binding CDR1” is a CDR1that binds, preferably specifically, to Staphylococcal PNAG/dPNAG, andis derived from either the heavy or light chain variable regions of theantibodies described herein or produced by hybridomas deposited underATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933. It may have anamino acid sequence selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19 and SEQ IDNO: 22. Similar respective definitions apply to StaphylococcalPNAG/dPNAG-binding CDR2 and CDR3.

A “Staphylococcal PNAG/dPNAG-binding CDR2” is a CDR2 that binds,preferably specifically, to Staphylococcal PNAG/dPNAG, and is derivedfrom either the heavy or light chain variable regions of the antibodiesdescribed herein or produced by the hybridomas deposited under ATCCAccession Nos. PTA-5931, PTA-5932 and PTA-5933. It may have an aminoacid sequence selected from the group consisting of SEQ ID NO: 8, SEQ IDNO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20 and SEQ ID NO: 23.

A “Staphylococcal PNAG/dPNAG-binding CDR3” is a CDR3 that binds,preferably specifically, to Staphylococcal PNAG/dPNAG, and is derivedfrom either the heavy or light chain variable regions of the antibodiesdescribed herein or produced by the hybridomas deposited under ATCCAccession Nos. PTA-5931, PTA-5932 and PTA-5933. It may have an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 21 and SEQ ID NO: 24.

In addition to the sequences listed above, the invention intends toembrace functionally equivalent variants of these sequences includingconservative substitution variants in either the amino acid ornucleotide sequence, as described in greater detail below.

The peptides of the invention, including but not limited to theopsonophagocytic antibodies discussed herein, are useful inter alia indiagnostic methods aimed at detecting, in a sample in or from a subject,the PNAG/dPNAG antigen or PNAG-expressing bacteria (such as but notlimited to Staphylococcal bacteria that express PNAG). At a minimum,peptides useful in these methods need only recognize and bind toPNAG/dPNAG (such as Staphylococcal PNAG/dPNAG) regardless of whetherthey also enhance opsonization and phagocytosis. In importantembodiments, the antibodies and fragments thereof bind to PNAG/dPNAGselectively. Accordingly, they need only possess one or more of the CDRsderived from the antibody clones described herein or produced by thehybridomas deposited under ATCC Accession Nos. PTA-5931, PTA-5932 andPTA-5933. In preferred embodiments, the peptides comprise aPNAG/dPNAG-binding CDR3, and even more preferably, the peptides comprisea heavy chain PNAG/dPNAG-binding CDR3. It is to be understood that notall of the CDRs are required in order to effect binding to PNAG/dPNAG.However, in some embodiments the peptides comprise all of the CDRs of agiven antibody clone disclosed herein or produced by hybridomasdeposited under ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933.

In addition, it should be understood that the invention also embracesthe exchange of CDRs between the variable regions provided herein.Preferably, a heavy chain CDR is exchanged with another heavy chainvariable region CDR, and likewise, a light chain CDR is exchanged withanother light chain variable region CDR.

The amino acid sequences of the CDRs of the variable chains disclosed inthe present invention are as follows:

SEQ Clone Chain CDR ID NO: Sequence F598 Hv CDR1  7 GYYWS F598 Hv CDR2 8 YIHYSRSTNSNPALKS F598 Hv CDR3  9 DTYYYDSGDYEDAFDI F598 Lt CDR1 10TLSSGHSNYAIA F598 Lt CDR2 11 VNRDGSHIRGD F598 Lt CDR3 12 QTWGAGIRV F628Hv CDR1 13 NYYWS F628 Hv CDR2 14 YIHYSGSTNSNPSLKS F628 Hv CDR3 15DTYYESSGHWFDGLDV F628 Lt CDR1 16 TLDSEHSRYTIA F628 Lt CDR2 17VKSDGSHSKGD F628 Lt CDR3 18 QTWGPGIRV F630 Hv CDR1 19 NFGIS F630 Hv CDR220 WVSTYNGRTNYAQKFRG F630 Hv CDR3 21 DYYETSGYAYDDFAI F630 Lt CDR1 22TLSSGHSTYAIA F630 Lt CDR2 23 VNSDGSHTKGD F630 Lt CDR3 24 QTWGPGIRV

The nucleotide sequences of the CDRs of the variable chains disclosed inthe present invention are as follows:

SEQ Clone Chain CDR ID NO: Sequence F598 Hv CDR1 31GGT TAC TAC TGG AGT TAT F598 Hv CDR2 32 ATT CAT TAT AGT AGGAGC ACC AAC TCC AAC CCC GCC CTC AAG AGT F598 Hv CDR3 33GAT ACC TAT TAC TAT GAT AGT GGT GAT TAT GAG GAT GCT TTT GAT ATT F598 LtCDR1 34 ACT CTG AGC AGT GGC CAC AGC AAC TAC GCC ATC GCT F598 Lt CDR2 35GTT AAC AGA GAT GGC AGC CAC ATC AGG GGG GAC F598 Lt CDR3 36CAG ACC TGG GGC GCT  GGC ATT CGA GTG F628 Hv CDR1 37 AAT TAC TAC TGG AGTF628 Hv CDR2 38 TAT ATC CAT TAT AGT  GGG AGC ACC AAC TCC AATCCA TCC CTC AAG AGT F628 Hv CDR3 39 GAT ACT TAC TAT GAA AGTAGT GGT CAT TGG TTC GAC GGT TTG GAC GTC F628 Lt CDR1 40ACT CTG GAC AGT GAA CAC AGC AGA TAC ACC ATC GCA F628 Lt CDR2 41GTT AAG AGT GAT GGC AGT CAC AGC AAG GGG GAC F628 Lt CDR3 42CAG ACT TGG GGC CCT   GGC ATT CGA GTG F630 Hv CDR1 43AAC TTT GGT ATC AGT F630 Hv CDR2 44 TGG GTC AGC ACT TAC AATGGT CGC ACA AAT TAT GCA CAG AAG TTC CGG GGC F630 Hv CDR3 45GAT TAC TAT GAG ACT   AGT GGT TAC GCC TAT  GAT GAT TTT GCG ATC F630 LtCDR1 46 ACT CTG AGC AGT GGG CAC AGC ACC TAC GCC ATC GCG F630 Lt CDR2 47GTC AAC AGT GAT GGC AGC CAC ACC AAG GGG GAC F630 Lt CDR3 48CAG ACG TGG GGC CCT   GGC ATT CGA GTG

The peptides may also comprise a Staphylococcal PNAG/dPNAG-bindingvariable region. A Staphylococcal PNAG/dPNAG-binding variable region isa variable region (preferably an antibody variable region as describedherein or as derived from hybridomas deposited under ATCC Accession Nos.PTA-5931, PTA-5932 and PTA-5933) that a) binds to both PNAG and dPNAG,b) binds to PNAG but not dPNAG (referred to herein as a PNAG-bindingvariable region), or c) binds to dPNAG but not PNAG (referred to hereinas a dPNAG-binding variable region), regardless of the PNAG/dPNAGsource.

The present invention provides at least six different variable regions,at least three of which are heavy chain variable regions and at leastthree of which are light chain variable regions. SEQ ID NO: 1 and SEQ IDNO: 25 correspond to the amino acid and nucleotide sequence of the heavychain variable region derived from antibody clone F598. SEQ ID NO: 2 andSEQ ID NO: 26 correspond to the amino acid and nucleotide sequence ofthe light chain variable region derived from antibody clone F598. SEQ IDNO: 3 and SEQ ID NO: 27 correspond to the amino acid and nucleotidesequence of the heavy chain variable region derived from antibody cloneF628. SEQ ID NO: 4 and SEQ ID NO: 28 correspond to the amino acid andnucleotide sequence of the light chain variable region derived fromantibody clone F628. SEQ ID NO: 5 and SEQ ID NO: 29 correspond to theamino acid and nucleotide sequence of the heavy chain variable regionderived from antibody clone F630. SEQ ID NO: 6 and SEQ ID NO: 30correspond to the amino acid and nucleotide sequence of the light chainvariable region derived from antibody clone F630.

It is to be understood that the nucleic acids or peptides of theinvention may be derived from the sequences provided herein or from thedeposited hybridomas. These sequences can be cloned (e.g., by PCR) andinserted into a vector and/or cells in order to produce peptidescorresponding to full length variable regions or fragments of fulllength variable regions, and antibodies comprising the variable regions.It is therefore possible to generate antibodies or fragments thereofthat comprise a combination of light and heavy chain variable regions.For example, an antibody of the invention may comprise the heavy chainvariable region from MAb F598 (or from the antibody produced by thedeposited F598 hybridoma) and the light chain variable region of F630(or from the antibody produced by the deposited F630 hybridoma). It isto be understood that any combination of heavy and light chain variableregions (as disclosed herein or as comprised in antibodies produced byhybridomas deposited under ATCC Accession Nos. PTA-5931, PTA-5932 andPTA-5933) can be used in the synthesis of an antibody or antibodyfragment according to the invention.

Accordingly, the invention embraces antibodies or antibody fragmentsthat are comprised of the following variable region combinations: SEQ IDNO:1 and SEQ ID NO:2; SEQ ID NO:1 and SEQ ID NO:4; SEQ ID NO:1 and SEQID NO:6; SEQ ID NO:3 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; SEQID NO:3 and SEQ ID NO:6; SEQ ID NO:5 and SEQ ID NO:2; SEQ ID NO:5 andSEQ ID NO:4; and SEQ ID NO:5 and SEQ ID NO:6.

Similarly, the invention embraces antibodies or antibody fragments thatare comprised of the following variable region combinations:

1. heavy chain variable region from hybridoma F598 having ATCC AccessionNo. PTA-5931 and light chain variable region from hybridoma F598 havingATCC Accession No. PTA-5931;

2. heavy chain variable region from hybridoma F598 having ATCC AccessionNo. PTA-5931 and light chain variable region from hybridoma F628 havingATCC Accession No. PTA-5932;

3. heavy chain variable region from hybridoma F598 having ATCC AccessionNo. PTA-5931 and light chain variable region from hybridoma F630 havingATCC Accession No. PTA-5933;

4. heavy chain variable region from hybridoma F628 having ATCC AccessionNo. PTA-5932 and light chain variable region from hybridoma F598 havingATCC Accession No. PTA-5931;

5. heavy chain variable region from hybridoma F628 having ATCC AccessionNo. PTA-5932 and light chain variable region from hybridoma F628 havingATCC Accession No. PTA-5932;

6. heavy chain variable region from hybridoma F628 having ATCC AccessionNo. PTA-5932 and light chain variable region from hybridoma F630 havingATCC Accession No. PTA-5933;

7. heavy chain variable region from hybridoma F630 having ATCC AccessionNo. PTA-5933 and light chain variable region from hybridoma F598 havingATCC Accession No. PTA-5931;

8. heavy chain variable region from hybridoma F630 having ATCC AccessionNo. PTA-5933 and light chain variable region from hybridoma F628 havingATCC Accession No. PTA-5932; and

9. heavy chain variable region from hybridoma F630 having ATCC AccessionNo. PTA-5933 and light chain variable region from hybridoma F630 havingATCC Accession No. PTA-5933.

The invention intends to capture antibody and antibody fragments ofvarious isotypes. The deposited hybridomas produce IgG2 isotypeantibodies. However, the recombined immunoglobulin (Ig) genes,particularly the variable region genes, can be isolated from thedeposited hybridomas, as described in the Examples, and cloned into anIg recombination vector that codes for human Ig constant region genes ofboth heavy and light chains. Using this technique, IgG1 isotypeantibodies that bind to Staphylococcal PNAG/dPNAG and thereby enhanceopsonophagocytosis of PNAG-expressing bacteria (such as Staphylococci)have been identified, synthesized and isolated.

The antibodies may be of an IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, IgA1,IgA2, or sIgA isotype. The invention intends to capture isotypes foundin non-human species as well such as but not limited to IgY in birds andsharks. Vectors encoding the constant regions of various isotypes areknown and previously described. (See, for example, Preston et al.Production and characterization of a set of mouse-human chimericimmunoglobulin G (IgG) subclass and IgA monoclonal antibodies withidentical variable regions specific for P. aeruginosa serogroup O6lipopolysaccharide. Infect Immun. 1998 September; 66(9):4137-42; Colomaet al. Novel vectors for the expression of antibody molecules usingvariable regions generated by polymerase chain reaction. J ImmunolMethods. 1992 Jul. 31; 152(1):89-104; Guttieri et al. Cassette vectorsfor conversion of Fab fragments into full-length human IgG1 monoclonalantibodies by expression in stably transformed insect cells. HybridHybridomics. 2003 June; 22(3):135-45; McLean et al. Human and murineimmunoglobulin expression vector cassettes. Mol Immunol. 2000 October;37(14):837-45; Walls et al. Vectors for the expression of PCR-amplifiedimmunoglobulin variable domains with human constant regions. NucleicAcids Res. 1993 Jun. 25; 21(12):2921-9; Norderhaug et al. Versatilevectors for transient and stable expression of recombinant antibodymolecules in mammalian cells. J Immunol Methods. 1997 May 12;204(1):77-87.)

As used herein, the term “peptide” includes monoclonal antibodies,functionally active and/or equivalent antibody fragments, andfunctionally active and/or equivalent peptides and polypeptides.

The peptides of the invention are isolated peptides. As used herein, theterm “isolated peptides” means that the peptides are substantially pureand are essentially free of other substances with which they may befound in nature or in vivo systems to an extent practical andappropriate for their intended use. In particular, the peptides aresufficiently pure and are sufficiently free from other biologicalconstituents of their hosts cells so as to be useful in, for example,producing pharmaceutical preparations or sequencing. Because an isolatedpeptide of the invention may be admixed with a pharmaceuticallyacceptable carrier in a pharmaceutical preparation, the peptide maycomprise only a small percentage by weight of the preparation. Thepeptide is nonetheless substantially pure in that it has beensubstantially separated from the substances with which it may beassociated in living systems.

The peptides of the invention bind to PNAG and/or dPNAG, preferably in aselective manner. As used herein, the terms “selective binding” and“specific binding” are used interchangeably to refer to the ability ofthe peptide to bind with greater affinity to PNAG and/or dPNAG andfragments thereof than to non-PNAG derived compounds. That is, peptidesthat bind selectively to PNAG and/or dPNAG will not bind to non-PNAGderived compounds to the same extent and with the same affinity as theybind to PNAG and/or dPNAG and fragments thereof, with the exception ofcross reactive antigens or molecules made to be mimics of PNAG/dPNAGsuch as peptide mimetics of carbohydrates or variable regions ofanti-idiotype antibodies that bind to the PNAG/dPNAG-binding peptides inthe same manner as PNAG/dPNAG. Antibodies that bind selectively to PNAGbind to PNAG with greater affinity than to dPNAG. Antibodies that bindto dPNAG may also bind to dPNAG with lesser, comparable or greateraffinity than to PNAG. In preferred embodiments, the peptides of theinvention bind solely to PNAG and/or dPNAG and fragments thereof, andeven more preferably, they at least bind to dPNAG. As used herein, abinding peptide that binds selectively or specifically to StaphylococcalPNAG/dPNAG may also bind PNAG/dPNAG from other sources and will bindwith lesser affinity (if at all) to non-PNAG/dPNAG derived compounds.Lesser affinity may include at least 10% less, 20% less, 30% less, 40%less, 50% less, 60% less, 70% less, 80% less, 90% less, or 95% less.Thus, “selective” in this sense refers to the binding to PNAG/dPNAGrather than to the Staphylococcus-derived form of PNAG/dPNAG.

As stated earlier, the invention provides peptides e.g., antibodies orantibody fragments, that bind to Staphylococcal PNAG and/or dPNAG. Suchantibodies preferably enhance opsonization and phagocytosis (i.e.,opsonophagocytosis) of PNAG-expressing bacteria (such as PNAG-expressingStaphylococci), and as a result are useful in the prevention and therapyof some forms of bacterial infections in a subject. Opsonization refersto a process by which phagocytosis is facilitated by the deposition ofopsonins (e.g., antibody and/or opsonic complement factors such as C4bor C3b or any other factor capable of promoting opsonophagocytosis) onthe antigen. Phagocytosis and opsonophagocytosis refer to the process bywhich phagocytic cells (e.g., macrophages, dendritic cells, andpolymorphonuclear leukocytes (PMNL)) engulf material and enclose itwithin a vacuole (e.g., a phagosome) in their cytoplasm. Thus,antibodies or antibody fragments that opsonize bacteria and enhancephagocytosis are antibodies or antibody fragments that recognize anddeposit onto an antigen, and in doing so, facilitate the uptake andengulfment of the antigen (and the antigen-bearing substance, e.g.,Staphylococcal bacteria) by phagocytic cells. Generally, in order toenhance phagocytosis and opsonization, the antibody comprises an Fcdomain or region. The Fc domain is recognized by Fc receptor bearingcells (e.g., antigen presenting cells such as macrophages, or PMNL). Asused herein, “to enhance opsonophagocytosis” means to increase thelikelihood that an antigen or an antigen bearing substrate will berecognized and engulfed by a phagocytic cell, via antibody deposition.This enhancement can be measured by reduction in bacterial load in vivoor by bacterial cell killing in vitro using the in vitro methodsdescribed below.

Opsonization assays are standard in the art. Generally such assaysmeasure the amount of bacterial killing in the presence of an antibody,an antigen (expressed on the target bacterial cell), complement, andphagocytic cells. Serum from either animals or humans is commonly usedas a source of complement, and polymorphonuclear cells from animals orhumans are commonly used as a source of phagocytic cells. The targetcell for opsonophagocytic killing can be prokaryotic (as in the presentinvention) or eukaryotic, depending upon which cell type expresses theantigen. Cell killing can be measured by viable cell counts prior to andfollowing incubation of the reaction components. Alternatively, cellkilling can be quantitated by measuring cell contents in the supernatantof the reaction mixture (e.g., release of radioactive chromium orrelease of intracellular enzymes such as lactate dehydrogenase). Otherassays will be apparent to those of skill in the art, having read thepresent specification, which are useful for determining whether anantibody or antibody fragment that binds to Staphylococcal PNAG and/ordPNAG also stimulates opsonization and phagocytosis.

The present invention provides, inter alia, PNAG/dPNAG-specific humanmonoclonal antibodies that enhance opsonic killing of PNAG-expressingbacteria such as but not limited to Staphylococci. These antibodies arenamed F598, F628 and F630. When used in vivo in humans, human monoclonalantibodies are far less likely to be immunogenic (as compared toantibodies from another species). As a result, these antibodiesrepresent novel agents useful in the design of vaccines as well aspassive immunotherapy targeting bacterial strains that express PNAG suchas but not limited to Staphylococci.

The synthesis of these monoclonal antibodies is described in theExamples. Briefly, the antibodies were derived as follows: B cells wereharvested from individuals recovering from a Staphylococcal infection.Harvested B cells were transformed using Epstein-Barr virus and, after aperiod of growth and screening for secretion of antibody to PNAG/dPNAG,fused with the immortalized human-mouse myeloma cell line partnerdesignated HMMA 2.5. After an initial period of growth of the fusedcells, single antibody producing clones were isolated, grown andanalyzed separately using a binding assay (e.g., ELISA). Threehybridomas were selected based on the ability of their secreted antibodyto bind to Staphylococcal PNAG and/or dPNAG. All three antibodies wereof the IgG2 isotype and were used as a source of antibody of the IgG2isotype. Variable regions were cloned from the hybridomas by PCR asdescribed above.

Variable region nucleic acids for the heavy and light chains of theantibodies were cloned into an human Ig expression vector (i.e., TCAE6)that contained the IgG1 (gamma 1) constant region coding sequences forthe heavy chain and the lambda constant region for the light chains.(See, for example, Preston et al. Production and characterization of aset of mouse-human chimeric immunoglobulin G (IgG) subclass and IgAmonoclonal antibodies with identical variable regions specific for P.aeruginosa serogroup O6 lipopolysaccharide. Infect Immun. 1998September; 66(9):4137-42.) The variable regions can be placed in anyvector that encodes constant region coding sequences. For example, humanIg heavy-chain constant-region expression vectors containing genomicclones of the human IgG2, IgG3, IgG4 and IgA heavy-chain constant-regiongenes and lacking variable-region genes have been described in Coloma,et al. 1992 J. Immunol. Methods 152:89-104.)

These expression vectors were then transfected into cells (e.g., CHODG44 cells), the cells were grown in vitro, and IgG1 was subsequentlyharvested from the supernatant. Resultant antibodies possessed humanvariable regions and human IgG1 and lambda constant regions. Theirability to bind to PNAG and/or dPNAG and to enhance opsonization andphagocytosis of PNAG-expressing bacteria such as Staphylococci wasevaluated using binding and opsonophagocytic killing assays such asthose described herein.

“Isolated antibodies” as used herein refer to antibodies that aresubstantially physically separated from other cellular material (e.g.,separated from cells which produce the antibodies) or from othermaterial that hinders their use either in the diagnostic or therapeuticmethods of the invention. Preferably, the isolated antibodies arepresent in a homogenous population of antibodies (e.g., a population ofmonoclonal antibodies). Compositions of isolated antibodies can howeverbe combined with other components such as but not limited topharmaceutically acceptable carriers, adjuvants, and the like.

“Isolated antibody producing cells” including isolated hybridomas andisolated recombinant cells (such as those described herein), as usedherein, refer to antibody-producing cells that are substantiallyphysically separated from other cells, other bodily material (e.g.,ascites tissue and fluid), and other material that hinders their use inthe production of, for example, an isolated and preferably homogenousantibody population. The hybridomas deposited with the ATCC under theBudapest Treaty as ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933on Apr. 21, 2004 are considered to be examples of isolated antibodyproducing cells and more specifically isolated hybridomas.

Thus in one embodiment, the peptide of the invention is an isolatedintact soluble monoclonal antibody specific for Staphylococcal PNAGand/or dPNAG. As used herein, the term “monoclonal antibody” refers to ahomogenous population of immunoglobulins that specifically bind to anidentical epitope (i.e., antigenic determinant). The peptide of theinvention in one embodiment is, for example, a monoclonal antibodyhaving a heavy chain variable region having an amino acid sequence ofSEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. The monoclonal antibody canhave a light chain variable region having an amino acid sequence of SEQID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Monoclonal antibodies having anycombination of light chain and heavy chain variable regions are embracedby the invention.

The invention intends to encompass antibodies other than, for example,clones F598, F628 and F630, provided that such antibodies have thebinding characteristics of the monoclonal antibodies described herein.Optionally, these additional antibodies also enhance opsonophagocytosisof PNAG-expressing bacterial strains such as but not limited toPNAG-expressing Staphylococci. One of ordinary skill in the art caneasily identify antibodies having the functional characteristics (e.g.,binding, opsonizing and phagocytosing attributes) of these monoclonalantibody using the screening and binding assays set forth in detailherein.

In other embodiments, the peptide is an antibody fragment. As iswell-known in the art, only a small portion of an antibody molecule, theparatope, is involved in the binding of the antibody to its epitope(see, in general, Clark, W. R. (1986) The Experimental Foundations ofModern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991)Essential Immunology, 7th Ed., Blackwell Scientific Publications,Oxford; and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology,Infection and Immunity (2004) 1^(st) Ed. American Society forMicrobiology Press, Washington D.C.). The pFc′ and Fc regions of theantibody, for example, are effectors of the complement cascade and canmediate binding to Fc receptors on phagocytic cells, but are notinvolved in antigen binding. An antibody from which the pFc′ region hasbeen enzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. An isolated F(ab′)₂ fragment isreferred to as a bivalent monoclonal fragment because of its two antigenbinding sites. Similarly, an antibody from which the Fc region has beenenzymatically cleaved, or which has been produced without the Fc region,designated an Fab fragment, retains one of the antigen binding sites ofan intact antibody molecule. Proceeding further, Fab fragments consistof a covalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd (heavy chain variable region). The Fd fragmentsare the major determinant of antibody specificity (a single Fd fragmentmay be associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

The terms Fab, Fc, pFc′, F(ab′)₂ and Fv are employed with eitherstandard immunological meanings [Klein, Immunology (John Wiley, NewYork, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations ofModern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991)Essential Immunology, 7th Ed., (Blackwell Scientific Publications,Oxford); and Pier G B, Lyczak J B, Wetzler L M, (eds). Immunology,Infection and Immunity (2004) 1^(st) Ed. American Society forMicrobiology Press, Washington D.C.].

In other embodiments, the Fc portions of the antibodies of the inventionmay be replaced so as to produce IgM as well as human IgG antibodiesbearing some or all of the CDRs of the monoclonal antibodies describedherein or produced by the hybridomas deposited under ATCC Accession Nos.PTA-5931, PTA-5932 and PTA-5933. Of particular importance is theinclusion of a Staphylococcal PNAG/dPNAG-binding CDR3 region and, to alesser extent, the other CDRs and portions of the framework regions ofthe monoclonal antibodies described herein or produced by the hybridomasdeposited under ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933.Such human antibodies will have particular clinical utility in that theywill recognize and bind, preferably selectively, to Staphylococcal PNAGand/or dPNAG, but will not evoke an immune response in humans againstthe antibody itself.

The invention also intends to include functionally equivalent variantsof the Staphylococcal PNAG/dPNAG-binding peptides. A “functionallyequivalent variant” is a compound having the same function (i.e., theability to bind to Staphylococcal PNAG and/or dPNAG and in someembodiments to facilitate opsonization of PNAG-expressing bacterialstrains) as the peptides of the invention. A functionally equivalentvariant may be peptide in nature but it is not so limited. For example,it may be a carbohydrate, a peptidomimetic, etc. In importantembodiments, the functionally equivalent variant is a peptide having theamino acid sequence of a variable region or a CDR with conservativesubstitutions therein, that is still capable of binding toStaphylococcal PNAG and/or dPNAG. An example of a functionallyequivalent variant of Staphylococcal PNAG/dPNAG-binding CDR3 from theheavy chain variable region of clone F598 (i.e., SEQ ID NO:1) is apeptide having conservative substitutions in SEQ ID NO:1 which bind,preferably specifically, to Staphylococcal PNAG and/or dPNAG, andoptionally which enhances opsonization of PNAG-expressing bacterialstrains such as PNAG-expressing Staphylococci. As used herein,“conservative substitution” refers to an amino acid substitution whichdoes not alter the relative charge or size characteristics of thepeptide in which the amino acid substitution is made. Conservativesubstitutions of amino acids include substitutions made amongst aminoacids with the following groups: (1) M,I,L,V; (2) F,Y,W; (3) K,R,H; (4)A,G; (5) S,T; (6) Q,N; and, (7) E,D.

Functional equivalent variants can have identity to the peptidesexplicitly recited herein. That is, such variants may have at least 99%identity, at least 98% identity, at least 97% identity, at least 96%identity, at least 95% identity, at least 94% identity, at least 93%identity, at least 92% identity, at least 91% identity, at least 90%identity, at least 85% identity, at least 80% identity, at least 75%identity, at least 70% identity, at least 65% identity, at least 60%identity, at least 55% identity, at least 50% identity, at least 45%identity, at least 40% identity, at least 35% identity, at least 30%identity, at least 25% identity, at least 20% identity, at least 10%identity, or at least 5% identity to the amino acid sequences providedherein.

Functional equivalence refers to an equivalent activity (e.g., bindingto Staphylococcal PNAG and/or dPNAG, or enhancing opsonophagocytosis ofPNAG-expressing bacteria such as PNAG-expressing Staphylococci), howeverit also embraces variation in the level of such activity. For example, afunctional equivalent is a variant that binds to Staphylococcal PNAGand/or dPNAG with lesser, equal, or greater affinity than the monoclonalantibody clones described herein, provided that the variant is stilluseful in the invention (i.e., it binds to Staphylococcal PNAG and/ordPNAG and optionally enhances opsonophagocytosis of PNAG-expressingbacteria such as PNAG-expressing Staphylococci).

Such substitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding the particular CDR ora peptide comprising the CDR amino acid sequences described herein.These and other methods for altering a CDR containing peptide will beknown to those of ordinary skill in the art and may be found inreferences which compile such methods, e.g. Sambrook or Ausubel, notedabove. In some embodiments, however, due to the size of the CDRs, it maybe more convenient to synthesize the variant peptides using a peptidesynthesizer such as those commercially available. The activity offunctionally equivalent variants of the StaphylococcalPNAG/dPNAG-binding CDR can be tested by the binding assays, and in somecases biological activity assays, discussed in more detail below. Asused herein, the terms “functional variant”, “functionally equivalentvariant” and “functionally active variant” are used interchangeably.

As used herein the term “functionally active antibody fragment” means afragment of an antibody molecule including a Staphylococcal PNAG-bindingor dPNAG-binding region of the invention which retains the ability tobind to Staphylococcal PNAG or dPNAG respectively, preferably in aspecific manner. Such fragments can be used both in vitro and in vivo.In particular, well-known functionally active antibody fragments includebut are not limited to F(ab′)₂, Fab, Fv and Fd fragments of antibodies.These fragments which lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). As another example, single-chain antibodies can be constructedin accordance with the methods described in U.S. Pat. No. 4,946,778 toLadner et al. Such single-chain antibodies include the variable regionsof the light and heavy chains joined by a flexible linker moiety.Methods for obtaining a single domain antibody (“Fd”) which comprises anisolated variable heavy chain single domain, also have been reported(see, for example, Ward et al., Nature 341:644-646 (1989), disclosing amethod of screening to identify an antibody heavy chain variable region(V_(H) single domain antibody) with sufficient affinity for its targetepitope to bind thereto in isolated form). Methods for makingrecombinant Fv fragments based on known antibody heavy chain and lightchain variable region sequences are known in the art and have beendescribed, e.g., Moore et al., U.S. Pat. No. 4,462,334. Other referencesdescribing the use and generation of antibody fragments include e.g.,Fab fragments (Tijssen, Practice and Theory of Enzyme Immunoassays(Elsevier, Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistry12: 1130 (1973); Sharon et al., Biochemistry 15: 1591 (1976); Ehrlich etal., U.S. Pat. No. 4,355,023) and portions of antibody molecules(Audilore-Hargreaves, U.S. Pat. No. 4,470,925). Thus, those skilled inthe art may construct antibody fragments from various portions of intactantibodies without destroying the specificity of the antibodies forStaphylococcal PNAG and/or dPNAG.

In important aspects of the invention, the functionally active antibodyfragment also retains the ability to opsonize and phagocytosePNAG-expressing bacteria such as PNAG-expressing Staphylococci. In thislatter instance, the antibody fragment includes an Fc region as well asan epitope binding domain. The Fc region allows the antibody fragment tobind to Fc receptor positive cells, which subsequently phagocytose theepitope bound by the Fab region of the antibody.

Additionally small peptides including those containing theStaphylococcal PNAG/dPNAG-binding CDR3 region may easily be synthesizedor produced by recombinant means to produce the peptide of theinvention. Such methods are well known to those of ordinary skill in theart. Peptides can be synthesized, for example, using automated peptidesynthesizers which are commercially available. The peptides can beproduced by recombinant techniques by incorporating the DNA expressingthe peptide into an expression vector and transforming cells with theexpression vector to produce the peptide.

Peptides, including antibodies, can be tested for their ability to bindto Staphylococcal PNAG and/or dPNAG using standard binding assays knownin the art. As an example of a suitable assay, PNAG and/or dPNAG, suchas Staphylococcal PNAG and/or dPNAG, can be immobilized on a surface(such as in a well of a multi-well plate) and then contacted with alabeled peptide. The amount of peptide that binds to the PNAG and/ordPNAG (and thus becomes itself immobilized onto the surface) may then bequantitated to determine whether a particular peptide binds to PNAGand/or dPNAG. Alternatively, the amount of peptide not bound to thesurface may also be measured. In a variation of this assay, the peptidecan be tested for its ability to bind directly to a PNAG-expressingcolony grown in vitro.

Peptide binding can also be tested using a competition assay. If thepeptide being tested (including an antibody) competes with themonoclonal antibodies or antibody fragments described herein, as shownby a decrease in binding of the monoclonal antibody or fragment, then itis likely that the peptide and the monoclonal antibody bind to the same,or at least an overlapping, epitope. In this assay system, the antibodyor antibody fragment is labeled and the PNAG and/or dPNAG is immobilizedonto the solid surface. These and other assays are described in moredetail herein. In this way, competing peptides including competingantibodies can be identified. The invention embraces peptides and inparticular antibodies (and fragments thereof) that compete with antibodyF598, F628 or F630 for binding to PNAG/dPNAG (i.e., antibodies thatrecognize and bind to the same epitopes as F598, F628 or F630).

Standard binding assays are well known in the art, and a number of theseare suitable in the present invention including ELISA, competitionbinding assay (as described above), sandwich assays, radioreceptorassays using radioactively labeled peptides or radiolabeled antibodies,immunoassays, etc. The nature of the assay is not essential provided itis sufficiently sensitive to detect binding of a small number ofpeptides.

A variety of other reagents also can be included in the binding mixture.These include reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalbinding. Such a reagent may also reduce non-specific or backgroundinteractions of the reaction components. Other reagents that improve theefficiency of the assay may also be used. The mixture of the foregoingassay materials is incubated under conditions under which the monoclonalantibody normally specifically binds PNAG and/or dPNAG such asStaphylococcal PNAG and/or dPNAG. Such conditions will preferably mimicphysiological conditions. The order of addition of components,incubation temperature, time of incubation, and other parameters of theassay may be readily determined. Such experimentation merely involvesoptimization of the assay parameters, not the fundamental composition ofthe assay. Incubation temperatures typically are between 4° C. and 40°C. Incubation times preferably are minimized to facilitate rapid, highthroughput screening, and typically are between 0.1 and 10 hours. Afterincubation, the presence or absence of specific binding between thepeptide and PNAG and/or dPNAG is detected by any convenient methodavailable to the user.

Typically, a plurality of assay mixtures are run in parallel withdifferent peptides or different peptide concentrations to obtain adifferent response to the various concentrations. One of theseconcentrations serves as a negative control, i.e., at zero concentrationof PNAG and/or dPNAG or at a concentration of PNAG and/or dPNAG belowthe limits of assay detection.

A separation step is often used to separate bound from unbound peptideor antibody. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components (e.g., peptide orantibody) is immobilized on a solid substrate via binding to PNAG and/ordPNAG. The unbound components may be easily separated from the boundfraction. The solid substrate can be made of a wide variety of materialsand in a wide variety of shapes, e.g., columns or gels ofpolyacrylamide, agarose or sepharose, microtiter plates, microbeads,resin particles, etc. The separation step preferably includes multiplerinses or washes. For example, when the solid substrate is a microtiterplate, the wells may be washed several times with a washing solution,which typically includes those components of the incubation mixture thatdo not participate in specific bindings such as salts, buffer,detergent, non-specific protein, etc. Where the solid substrate is amagnetic bead, the beads may be washed one or more times with a washingsolution and isolated using a magnet.

The peptides can be used alone or in conjugates with other moleculessuch as detection or cytotoxic agents in the detection and treatmentmethods of the invention, as described in more detail herein.

Typically, one of the components usually comprises, or is coupled orconjugated to a detectable label. A detectable label is a moiety, thepresence of which can be ascertained directly or indirectly. Generally,detection of the label involves an emission of energy by the label. Thelabel can be detected directly by its ability to emit and/or absorbphotons or other atomic particles of a particular wavelength (e.g.,radioactivity, luminescence, optical or electron density, etc.). A labelcan be detected indirectly by its ability to bind, recruit and, in somecases, cleave another moiety which itself may emit or absorb light of aparticular wavelength (e.g., epitope tag such as the FLAG epitope,enzyme tag such as horseradish peroxidase, etc.). An example of indirectdetection is the use of a first enzyme label which cleaves a substrateinto visible products. The label may be of a chemical, peptide ornucleic acid molecule nature although it is not so limited. Otherdetectable labels include radioactive isotopes such as P³² or H³,luminescent markers such as fluorochromes, optical or electron densitymarkers, etc., or epitope tags such as the FLAG epitope or the HAepitope, biotin, avidin, and enzyme tags such as horseradish peroxidase,β-galactosidase, etc. The label may be bound to a peptide during orfollowing its synthesis. There are many different labels and methods oflabeling known to those of ordinary skill in the art. Examples of thetypes of labels that can be used in the present invention includeenzymes, radioisotopes, fluorescent compounds, colloidal metals,chemiluminescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for thepeptides described herein, or will be able to ascertain such, usingroutine experimentation. Furthermore, the coupling or conjugation ofthese labels to the peptides of the invention can be performed usingstandard techniques common to those of ordinary skill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the peptides to low molecular weight haptens. Thesehaptens can then be specifically altered by means of a second reaction.For example, it is common to use haptens such as biotin, which reactswith avidin, or dinitrophenol, pyridoxal, or fluorescein, which canreact with specific anti-hapten antibodies.

Conjugation of the peptides including antibodies or fragments thereof toa detectable label facilitates, among other things, the use of suchagents in diagnostic assays. Another category of detectable labelsincludes diagnostic and imaging labels (generally referred to as in vivodetectable labels) such as for example magnetic resonance imaging (MRI):Gd(DOTA); for nuclear medicine: ²⁰¹Tl, gamma-emitting radionuclide99mTc; for positron-emission tomography (PET): positron-emittingisotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 111In.

The conjugations or modifications described herein employ routinechemistry, which chemistry does not form a part of the invention andwhich chemistry is well known to those skilled in the art of chemistry.The use of protecting groups and known linkers such as mono- andhetero-bifunctional linkers are well documented in the literature andwill not be repeated here.

As used herein, “conjugated” means two entities stably bound to oneanother by any physiochemical means. It is important that the nature ofthe attachment is such that it does not impair substantially theeffectiveness of either entity. Keeping these parameters in mind, anycovalent or non-covalent linkage known to those of ordinary skill in theart may be employed. In some embodiments, covalent linkage is preferred.Noncovalent conjugation includes hydrophobic interactions, ionicinteractions, high affinity interactions such as biotin-avidin andbiotin-streptavidin complexation and other affinity interactions. Suchmeans and methods of attachment are well known to those of ordinaryskill in the art.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The monoclonal antibodies described herein can also be used to produceanti-idiotypic antibodies that can be used to screen and identify otherantibodies having the same binding specificity as the monoclonalantibodies of the invention. An anti-idiotypic antibody is an antibodywhich recognizes unique determinants present on a monoclonal antibody ofthe invention. These determinants are located in the hypervariableregion of the antibody. It is this region that binds to a given epitopeand is thereby responsible for the specificity of the antibody. Suchanti-idiotypic antibodies can be produced using well-known hybridomatechniques (Kohler and Milstein, Nature, 256:495, 1975). As an example,an anti-idiotypic antibody can be prepared by immunizing a subject withthe monoclonal antibody. The immunized subject will recognize andrespond to the idiotypic determinants of the immunizing monoclonalantibody and produce an antibody to these idiotypic determinants. Byusing the anti-idiotypic antibodies of the immunized animal, which arespecific for the monoclonal antibody of the invention, it is possible toidentify other clones with the same idiotype as the monoclonal antibodyused for immunization. Idiotypic identity between monoclonal antibodiesof two cell lines demonstrates that the two monoclonal antibodies arethe same with respect to their recognition of the same epitopicdeterminant. Thus, by using anti-idiotypic antibodies, it is possible toidentify other hybridomas expressing monoclonal antibodies having thesame epitopic specificity. The invention intends to embrace all thefore-going antibody types.

The anti-idiotypic antibodies can also be used for active immunization(Herlyn, et al., Science, 232:100, 1986), since it is possible to usethe anti-idiotype technology to produce monoclonal antibodies that mimican epitope. For example, an anti-idiotypic monoclonal antibody made to afirst monoclonal antibody will have a binding domain in thehypervariable region which is the image of the epitope bound by thefirst monoclonal antibody. Thus, the anti-idiotypic monoclonal antibodycan be used for immunization, since the anti-idiotype monoclonalantibody binding domain effectively acts as an antigen.

The invention further contemplates bi-specific antibodies that include afirst antigen-binding domain specific for PNAG/dPNAG and a secondantigen binding domain specific for another moiety. The first antigenbinding domain specific for PNAG/dPNAG may comprise any of thePNAG/dPNAG binding peptides (including CDRs, variable regions, Fabfragments described herein or produced or derived from depositedhybridomas having ATCC Accession Nos. PTA-5931, PTA-5932 and PTA-5933).The second antigen binding domain may be specific for a moiety on a cellsuch as a bacterial cell or a host cell. Host cells may be immune systemcells or cells from the infected tissue. Antibodies for cell surfacemolecules expressed by immune system cells or from various host tissuecells are generally commercially available from sources such as Sigma orBD Biosciences Pharmingen. Those of ordinary skill in the art will beable to generate such bi-specific antibodies based on the teachingherein and the knowledge in the art. In a similar manner, the inventioncontemplates tri-specific antibodies also. (See, for example, U.S. Pat.Nos. 5,945,311 and 6,551,592 for bi-specific and tri-specific antibodygeneration.)

The sequences responsible for the specificity of the monoclonalantibodies of the invention have been determined. Accordingly, peptidesaccording to the invention can be prepared using recombinant DNAtechnology. There are entities in the United States which will performthis function commercially, such as Thomas Jefferson University and theScripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla,Calif.). For example, the variable region cDNA can be prepared bypolymerase chain reaction from the deposited hybridoma RNA usingdegenerate or non-degenerate primers (derived from the amino acidsequence). The cDNA can be subcloned to produce sufficient quantities ofdouble stranded DNA for sequencing by conventional sequencing reactionsor equipment.

With knowledge of the nucleic acid sequences of the heavy chain andlight chain variable domains of the anti-Staphylococcal PNAG/dPNAGmonoclonal antibody, one of ordinary skill in the art is able to producenucleic acids which encode this antibody or which encode the variousantibody fragments, humanized antibodies, or polypeptides describedabove. It is contemplated that such nucleic acids will be operablyjoined to other nucleic acids forming a recombinant vector for cloningor for expression of the peptides of the invention. The presentinvention includes any recombinant vector containing the codingsequences, or part thereof, whether for prokaryotic or eukaryotictransformation, transfection or gene therapy. Such vectors may beprepared using conventional molecular biology techniques, known to thosewith skill in the art, and would comprise DNA coding sequences for theCDR region (and preferably the CDR3 region) and additional variablesequences contributing to the specificity of the antibodies or partsthereof, as well as other non-specific peptide sequences and a suitablepromoter either with (Whittle et al., Protein Eng. 1:499, 1987 andBurton et al., Science 266:1024-1027, 1994) or without (Marasco et al.,Proc. Natl. Acad. Sci. (USA) 90:7889, 1993 and Duan et al., Proc. Natl.Acad. Sci. (USA) 91:5075-5079, 1994) a signal sequence for export orsecretion. Such vectors may be transformed or transfected intoprokaryotic (Huse et al., Science 246:1275, 1989, Ward et al., Nature341: 644-646, 1989; Marks et al., J. Mol. Biol. 222:581, 1991 and Barbaset al., Proc. Natl. Acad. Sci. (USA) 88:7978, 991) or eukaryotic(Whittle et al., 1987 and Burton et al., 1994) cells or used for genetherapy (Marasco et al., 1993 and Duan et al., 1994) by conventionaltechniques, known to those with skill in the art.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

The expression vectors of the present invention include regulatorysequences operably joined to a nucleotide sequence encoding one of thepeptides of the invention. As used herein, the term “regulatorysequences” means nucleotide sequences which are necessary for, orconducive to, the transcription of a nucleotide sequence which encodes adesired polypeptide and/or which are necessary for or conducive to thetranslation of the resulting transcript into the desired polypeptide.Regulatory sequences include, but are not limited to, 5′ sequences suchas operators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals. The vectors of the invention mayoptionally include 5′ leader or signal sequences, 5′ or 3′ sequencesencoding fusion products to aid in protein purification, and variousmarkers which aid in the identification or selection of transformants.The choice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art. The subsequentpurification of the peptides may be accomplished by any of a variety ofstandard means known in the art.

A preferred vector for screening peptides, but not necessarily preferredfor the mass production of the peptides of the invention, is arecombinant DNA molecule containing a nucleotide sequence that codes forand is capable of expressing a fusion polypeptide containing, in thedirection of amino- to carboxy-terminus, (1) a prokaryotic secretionsignal domain, (2) a polypeptide of the invention, and, optionally, (3)a fusion protein domain. The vector includes DNA regulatory sequencesfor expressing the fusion polypeptide, preferably prokaryotic regulatorysequences. Such vectors can be constructed by those with skill in theart and have been described by Smith et al. (Science 228:1315-1317,1985), Clackson et al. (Nature 352:624-628, 1991); Kang et al. (in“Methods: A Companion to Methods in Enzymology: Vol. 2”, R. A. Lernerand D. R. Burton, ed. Academic Press, NY, pp 111-118, 1991); Barbas etal. (Proc. Natl. Acad. Sci. (USA) 88:7978-7982, 1991), Roberts et al.(Proc. Natl. Acad. Sci. (USA) 89:2429-2433, 1992)

A fusion polypeptide may be useful for purification of the peptides ofthe invention. The fusion domain may, for example, include a poly-Histail which allows for purification on Ni+ columns or the maltose bindingprotein of the commercially available vector pMAL (New England BioLabs,Beverly, Mass.). A currently preferred, but by no means necessary,fusion domain is a filamentous phage membrane anchor. This domain isparticularly useful for screening phage display libraries of monoclonalantibodies but may be of less utility for the mass production ofantibodies. The filamentous phage membrane anchor is preferably a domainof the cpIII or cpVIII coat protein capable of associating with thematrix of a filamentous phage particle, thereby incorporating the fusionpolypeptide onto the phage surface, to enable solid phase binding tospecific antigens or epitopes and thereby allow enrichment and selectionof the specific antibodies or fragments encoded by the phagemid vector.

The secretion signal is a leader peptide domain of a protein thattargets the protein membrane of the host cell, such as the periplasmicmembrane of gram negative bacteria. A preferred secretion signal for E.coli is a pelB secretion signal. The predicted amino acid residuesequences of the secretion signal domain from two pelB gene producingvariants from Erwinia carotova are described in Lei, et al. (Nature381:543-546, 1988). The leader sequence of the pelB protein haspreviously been used as a secretion signal for fusion proteins (Better,et al., Science 240:1041-1043, 1988; Sastry, et al., Proc. Natl. Acad.Sci (USA) 86:5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad.Sci. (USA) 87:8095-8099, 1990). Amino acid residue sequences for othersecretion signal polypeptide domains from E. coli useful in thisinvention can be found in Oliver, In Neidhard, F. C. (ed.), Escherichiacoli and Salmonella Typhimurium, American Society for Microbiology,Washington, D.C., 1:56-69 (1987).

To achieve high levels of gene expression in E. coli, it is necessary touse not only strong promoters to generate large quantities of mRNA, butalso ribosome binding sites to ensure that the mRNA is efficientlytranslated. In E. coli, the ribosome binding site includes an initiationcodon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotidesupstream from the initiation codon (Shine, et al., Nature 254:34, 1975).The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence,is complementary to the 3′ end of E. coli 16S rRNA. Binding of theribosome to mRNA and the sequence at the 3′ end of the mRNA can beaffected by several factors: (i) the degree of complementarity betweenthe SD sequence and 3′ end of the 16S rRNA; (ii) the spacing andpossibly the DNA sequence lying between the SD sequence and the AUG(Roberts, et al., Proc. Natl. Acad. Sci. (USA) 76:760, 1979a: Roberts,et al., Proc. Natl. Acad. Sci. (USA) 76:5596, 1979b; Guarente, et al.,Science 209:1428, 1980; and Guarente, et al., Cell 20:543, 1980).Optimization is achieved by measuring the level of expression of genesin plasmids in which this spacing is systematically altered. Comparisonof different mRNAs shows that there are statistically preferredsequences from positions −20 to +13 (where the A of the AUG is position0) (Gold, et al., Annu. Rev. Microbiol. 35:365, 1981). Leader sequenceshave been shown to influence translation dramatically (Roberts, et al.,1979a, b supra); and (iii) the nucleotide sequence following the AUG,which affects ribosome binding (Taniguchi, et al., J. Mol. Biol.,118:533, 1978).

The 3′ regulatory sequences define at least one termination (stop) codonin frame with and operably joined to the heterologous fusionpolypeptide.

In preferred embodiments with a prokaryotic expression host, the vectorutilized includes a prokaryotic origin of replication or replicon, i.e.,a DNA sequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extra-chromosomally in aprokaryotic host cell, such as a bacterial host cell, transformedtherewith. Such origins of replication are well known in the art.Preferred origins of replication are those that are efficient in thehost organism. A preferred host cell is E. coli. For use of a vector inE. coli, a preferred origin of replication is ColE1 found in pBR322 anda variety of other common plasmids. Also preferred is the p15A origin ofreplication found on pACYC and its derivatives. The ColE1 and p15Areplicons have been extensively utilized in molecular biology, areavailable on a variety of plasmids and are described by Sambrook. etal., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory Press, 1989).

In addition, those embodiments that include a prokaryotic repliconpreferably also include a gene whose expression confers a selectiveadvantage, such as drug resistance, to a bacterial host transformedtherewith. Typical bacterial drug resistance genes are those that conferresistance to ampicillin, tetracycline, neomycin/kanamycin orchloramphenicol. Vectors typically also contain convenient restrictionsites for insertion of translatable DNA sequences. Exemplary vectors arethe plasmids pUC18 and pUC19 and derived vectors such as pcDNAIIavailable from Invitrogen (San Diego, Calif.).

When the peptide of the invention is an antibody including both heavychain and light chain sequences, these sequences may be encoded onseparate vectors or, more conveniently, may be expressed by a singlevector. The heavy and light chain may, after translation or aftersecretion, form the heterodimeric structure of natural antibodymolecules. Such a heterodimeric antibody may or may not be stabilized bydisulfide bonds between the heavy and light chains.

A vector for expression of heterodimeric antibodies, such as the intactantibodies of the invention or the F(ab′)₂, Fab or Fv fragmentantibodies of the invention, is a recombinant DNA molecule adapted forreceiving and expressing translatable first and second DNA sequences.That is, a DNA expression vector for expressing a heterodimeric antibodyprovides a system for independently cloning (inserting) the twotranslatable DNA sequences into two separate cassettes present in thevector, to form two separate cistrons for expressing the first andsecond polypeptides of a heterodimeric antibody. The DNA expressionvector for expressing two cistrons is referred to as a dicistronicexpression vector.

Preferably, the vector comprises a first cassette that includes upstreamand downstream DNA regulatory sequences operably joined via a sequenceof nucleotides adapted for directional ligation to an insert DNA. Theupstream translatable sequence preferably encodes the secretion signalas described above. The cassette includes DNA regulatory sequences forexpressing the first antibody polypeptide that is produced when aninsert translatable DNA sequence (insert DNA) is directionally insertedinto the cassette via the sequence of nucleotides adapted fordirectional ligation.

The dicistronic expression vector also contains a second cassette forexpressing the second antibody polypeptide. The second cassette includesa second translatable DNA sequence that preferably encodes a secretionsignal, as described above, operably joined at its 3′ terminus via asequence of nucleotides adapted for directional ligation to a downstreamDNA sequence of the vector that typically defines at least one stopcodon in the reading frame of the cassette. The second translatable DNAsequence is operably joined at its 5′ terminus to DNA regulatorysequences forming the 5′ elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion polypeptide comprising a secretion signal with apolypeptide coded by the insert DNA.

The peptides of the present invention may also be produced by eukaryoticcells such as CHO cells, human hybridomas, immortalized B-lymphoblastoidcells, and the like. In this case, a vector is constructed in whicheukaryotic regulatory sequences are operably joined to the nucleotidesequences encoding the peptide. The design and selection of anappropriate eukaryotic vector is within the ability and discretion ofone of ordinary skill in the art. The subsequent purification of thepeptides may be accomplished by any of a variety of standard means knownin the art.

In another embodiment, the present invention provides host cells, bothprokaryotic and eukaryotic, transformed or transfected with, andtherefore including, the vectors of the present invention.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences, as desired.

The invention also intends to embrace the use of the peptides describedherein in in vivo and in vitro methods. The methods of the invention areuseful for diagnosing as well as treating infections by PNAG-expressingbacteria such as Staphylococcal infections. “Staphylococci” as usedherein refers to all Staphylococcal bacterial species expressing thePNAG antigen. Bacteria that are classified as Staphylococci are wellknown to those of skill in the art and are described in the microbiologyliterature. Staphylococci expressing PNAG include but are not limited toStaphylococcus epidermidis (including RP62A (ATCC Number 35984), RP12(ATCC Number 35983), and M187), Staphylococcus aureus (including RN4220(pCN27) and MN8 mucoid), and strains such as Staphylococcus carnosustransformed with the genes in the ica locus (including TM300 (pCN27)).Other bacterial strains expressing PNAG naturally carry or aretransformed with the pga locus. Examples include E. coli, Yersiniapestis (Y. pestis), Y. entercolitica, Xanthomonas axonopodis,Pseudomonas fluorescens (all of which are sequenced species withcomplete pgaABCD loci), and Actinobacillus actinomycetemcomitans (A.actinomycetemcomitans), A. pleuropneumoniae, Ralstonia solanacearum(e.g., megaplasmid form), Bordetella pertussis (B. pertussis), B.parapertussis and B. bronchiseptica. Other bacterial strains expressingPNAG can be identified easily by those of ordinary skill in the art. Forinstance, bacteria that carry the ica or pga locus can produce PNAG. Oneof ordinary skill in the art can easily screen for the expression ofmRNA or protein related to the ica or pga locus since the nucleic acidsequences of the ica and pga locus are known (described in Heilmann, C.,O. Schweitzer, C. Gerke, N. Vanittanakom, D. Mack and F. Gotz (1996)Molecular basis of intercellular adhesion in the biofilm-formingStaphylococcus epidermidis. Molec. Microbiol. 20:1083 for S. epidermidisand in Cramton S E, Gerke C, Schnell N F, Nichols W W, Gotz F. Theintercellular adhesion (ica) locus is present in Staphylococcus aureusand is required for biofilm formation. Infect Immun. 1999 October;67(10):5427-33) for S. aureus; Blattner, F. R., G. Plunkett III, C. A.Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D.Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A.Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. Thecomplete genome sequence of Escherichia coli K-12. Science277:1453-1474. The genes reported by Blattner et al. were designatedycdSRQP in and renamed pgaABCD in Wang et al., J. Bacteriology, May2004, p. 2724-2734, Vol. 186, No. 9.) Additionally the capsule ofbacterial strains can be isolated and analyzed using liquidchromatography and mass spectroscopy.

The detection or diagnosis methods provided by the invention generallyinvolve contacting one or more peptides of the invention with a samplein or from a subject. Preferably, the sample is first harvested from thesubject, although in vivo detection methods are also envisioned. Thesample may include any body tissue or fluid that is suspected ofharboring the bacteria. For example, a Staphylococcal infection canoccur in essentially all tissues, organs and fluids of the human bodybut are most commonly found infecting the skin, bones, joints lungs andblood. An E. coli infection can occur, for example, in thegenito-urinary tract, as well as other tissues and locations. Y. pestisinfection is the cause of bubonic plague in the skin and pneumonicplague in the lung. B. pertussis infection causes whopping cough in therespiratory tract. Essentially any bodily fluid, tissue or organ such asskin, bone, joints, lungs, mucous such as phlegm and blood can be testedfor the presence of the bacteria.

As used herein, the term “treatment” refers to the administration ofpeptides to a subject for the purpose of achieving a medically desirablebenefit. Accordingly, “treatment” intends to embrace both “prophylactic”and “therapeutic” treatment methods. Prophylactic treatment methodsrefer to treatment administered to a subject prior to the diagnosis ofan infection (such as a Staphylococcal infection). In other words, thesubject does not present with symptoms of an infection (such as aStaphylococcal infection) although the subject may be at risk thereof.Therapeutic treatment methods refer to treatment administered to asubject after the diagnosis of an infection (such as a Staphylococcalinfection). In other words, the subject has been diagnosed as having aninfection (such as a Staphylococcal infection) or alternatively, thesubject may exhibit symptoms associated with such an infection.

The anti-PNAG/dPNAG antibodies of the invention are useful for inducingpassive immunization in a subject to prevent or limit the development ofsystemic infection and disease in those subjects at risk of exposure toinfectious agents. The method for inducing passive immunity to infectionby PNAG-expressing bacteria, such as Staphylococci such as S. aureus, isperformed by administering to a subject an effective amount of ananti-PNAG/dPNAG antibody (e.g., one that causes opsonization ofStaphylococci such as S. aureus). “Passive immunity” as used hereininvolves the administration of antibodies to a subject, wherein theantibodies are produced in a different subject (including subjects ofthe same and different species), such that the antibodies attach to thesurface of the bacteria and cause the bacteria to be phagocytosed.

The anti-PNAG/dPNAG antibody may be administered to any subject at riskof developing an infection by bacteria that express PNAG (e.g.,PNAG-expressing Staphylococcal infection) to induce passive immunity,and in some embodiments may be particularly suited for subjectsincapable of inducing active immunity to PNAG and/or dPNAG. A subjectthat is incapable of inducing an immune response is an immunocompromisedsubject (e.g. patient undergoing chemotherapy, AIDS patient, etc.) or asubject that has not yet developed an immune system (e.g. pre-termneonate).

A “subject” as used herein is a warm-blooded mammal and includes but isnot limited to humans, primates, agricultural animals such as horses,cows, swine, goats, sheep and chicken, and domestic animals such as dogsand cats. In some embodiments, the subject is a non-rodent subject. Anon-rodent subject is any subject as defined above, but specificallyexcluding rodents such as mice, rats, and rabbits. In some embodiments,the preferred subject is a human. In some instances, the subject may beone that has or will receive a prosthesis such as a hip or kneereplacement since such devices are especially prone to colonization bybacteria. As stated herein, some aspects of the invention provide fordetection and treatment of infections in plants also.

The anti-PNAG/dPNAG antibody of the invention is administered to thesubject in an effective amount for inducing immunity to PNAG-expressingbacteria (e.g., Staphylococci such as S. aureus). An “effective amountfor inducing immunity to PNAG-expressing bacteria” is an amount ofanti-PNAG/dPNAG antibody that is sufficient to (i) prevent infection bysuch bacteria from occurring in a subject that is exposed to suchbacteria; (ii) inhibit the development of infection, i.e., arresting orslowing its development; and/or (iii) relieve the effects of theinfection, i.e., reduction in bacterial load or complete eradication ofthe bacteria in infected subjects. As an example, an “effective amountfor inducing immunity to Staphylococci” as used herein is an amount ofanti-PNAG/dPNAG antibody that is sufficient to (i) prevent infection byStaphylococci from occurring in a subject that is exposed toStaphylococci; (ii) inhibit the development of infection, i.e.,arresting or slowing its development; and/or (iii) relieve the effectsof the infection, i.e., reduction in bacterial load or completeeradication of the bacteria in infected subjects.

Using routine procedures known to those of ordinary skill in the art,one can determine whether an amount of anti-PNAG/dPNAG antibody is an“effective amount for inducing immunity to infection” by using an invitro opsonization assay which is predictive of the degree ofopsonization of an antibody. An antibody that opsonizes PNAG-expressingbacteria such as PNAG-expressing Staphylococcal bacteria is one thatwhen added to a sample of such bacteria causes phagocytosis of thebacteria. An opsonization assay may be a colorimetric assay, achemiluminescent assay, a fluorescent or radiolabel uptake assay, a cellmediated bactericidal assay or other assay which measures the opsonicpotential of a material.

Antibody doses ranging from 1 ng/kg to 100 mg/kg, depending upon themode of administration, will be effective. The preferred range isbelieved to be between 500 ng and 500 μg/kg, and most preferably between1-100 μg/kg. The absolute amount will depend upon a variety of factorsincluding whether the administration is performed on a high risk subjectnot yet infected with the bacteria or on a subject already having aninfection, the concurrent treatment, the number of doses and theindividual patient parameters including age, physical condition, sizeand weight. These are factors well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.It is preferred generally that a maximum dose be used, that is, thehighest safe dose according to sound medical judgment.

Multiple doses of the antibodies of the invention are also contemplated.Generally immunization schemes involve the administration of a high doseof an antibody followed by subsequent lower doses of antibody after awaiting period of several weeks. Further doses may be administered aswell. The dosage schedule for passive immunization may require morefrequent administration. Desired time intervals for delivery of multipledoses of a particular PNAG/dPNAG antibody can be determined by one ofordinary skill in the art employing no more than routineexperimentation.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular anti-PNAG/dPNAGantibody selected, the particular condition being treated and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of protection without causing clinically unacceptable adverseeffects. Preferred modes of administration are parenteral routes. Theterm “parenteral” includes subcutaneous, intravenous, intramuscular,intraperitoneal, and intrasternal injection, or infusion techniques.Other routes include but are not limited to oral, nasal, dermal,sublingual, and local.

The anti-PNAG/dPNAG antibodies of the invention may be delivered inconjunction with other anti-bacterial drugs (e.g., antibiotics) or withother anti-bacterial antibodies. The use of antibiotics in the treatmentof bacterial infection is routine. A common administration vehicle(e.g., tablet, implant, injectable solution, etc.) may contain both theantibody of the invention and the anti-bacterial drug and/or antibody.Alternatively, the anti-bacterial drug and/or antibody can be separatelydosed. The anti-bacterial drug or antibody can also be conjugated to theanti-PNAG/dPNAG antibody.

Anti-bacterial drugs are well known and include: penicillin G,penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin,epicillin, hetacillin, pivampicillin, methicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, ticarcillin,avlocillin, mezlocillin, piperacillin, amdinocillin, cephalexin,cephradine, cefadoxil, cefaclor, cefazolin, cefuroxime axetil,cefamandole, cefonicid, cefoxitin, cefotaxime, ceftizoxime, cefmenoxine,ceftriaxone, moxalactam, cefotetan, cefoperazone, ceftazidme, imipenem,clavulanate, timentin, sulbactam, neomycin, erythromycin, metronidazole,chloramphenicol, clindamycin, lincomycin, vancomycin,trimethoprim-sulfamethoxazole, aminoglycosides, quinolones,tetracyclines and rifampin. (See Goodman and Gilman's, PharmacologicalBasics of Therapeutics, 8th Ed., 1993, McGraw Hill Inc.).

According to the methods of the invention, the peptide may beadministered in a pharmaceutical composition. In general, apharmaceutical composition comprises the peptide of the invention and apharmaceutically-acceptable carrier. Pharmaceutically-acceptablecarriers for peptides, monoclonal antibodies, and antibody fragments arewell-known to those of ordinary skill in the art. As used herein, apharmaceutically-acceptable carrier means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients, e.g., the ability of the peptide to bind toStaphylococcal PNAG and/or dPNAG and optionally to enhance opsonizationand phagocytosis.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials which arewell-known in the art. Exemplary pharmaceutically acceptable carriersfor peptides in particular are described in U.S. Pat. No. 5,211,657.Such preparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The peptides of the invention may be formulated into preparations insolid, semi-solid, liquid or gaseous forms such as tablets, capsules,powders, granules, ointments, solutions, depositories, inhalants andinjections, and usual ways for oral, parenteral or surgicaladministration. The invention also embraces pharmaceutical compositionswhich are formulated for local administration, such as by implants.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

When the compounds described herein (including peptide and non-peptidevarieties) are used therapeutically, in certain embodiments a desirableroute of administration may be by pulmonary aerosol. Techniques forpreparing aerosol delivery systems containing compounds are well knownto those of skill in the art. Generally, such systems should utilizecomponents which will not significantly impair the biological propertiesof the peptides (see, for example, Sciarra and Cutie, “Aerosols,” inRemington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;incorporated by reference). Those of skill in the art can readilydetermine the various parameters and conditions for producing aerosolswithout resort to undue experimentation.

The methods of the invention also encompass the step of administeringthe peptides of the invention in conjunction with conventional therapiesfor treating the underlying bacterial infection. For example, the methodof the invention may be practiced simultaneously with a conventionaltreatment, such as for example antibiotic therapy. In some embodiments,the peptides may be administered substantially simultaneously with theconventional treatment. By substantially simultaneously, it is meantthat a peptide of the invention is administered to a subject closeenough in time with the administration of the conventional treatment(e.g., antibiotic), whereby the two compounds may exert an additive oreven synergistic effect. In some instances, the peptide and the agent ofthe conventional treatment are conjugated to each other. In others, thecompounds are physically separate.

The peptides of the invention may be administered directly to a tissue.Preferably, the tissue is one in which the bacterial infection exists.Alternatively, the tissue is one in which the infection is likely toarise. Direct tissue administration may be achieved by direct injection.The peptides may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the peptides may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

In yet other embodiments, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT InternationalApplication No. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, claiming priority to U.S. patentapplication Ser. No. 213,668, filed Mar. 15, 1994). PCT/US/0307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining a biological macromolecule. The polymeric matrix may be usedto achieve sustained release of the agent in a subject. In accordancewith one aspect of the instant invention, the agent described herein maybe encapsulated or dispersed within the biocompatible, preferablybiodegradable polymeric matrix disclosed in PCT/US/03307. The polymericmatrix preferably is in the form of a microparticle such as amicrosphere (wherein the agent is dispersed throughout a solid polymericmatrix) or a microcapsule (wherein the agent is stored in the core of apolymeric shell). Other forms of the polymeric matrix for containing theagent include films, coatings, gels, implants, and stents. The size andcomposition of the polymeric matrix device is selected to result infavorable release kinetics in the tissue into which the matrix device isimplanted. The size of the polymeric matrix device further is selectedaccording to the method of delivery which is to be used, typicallyinjection into a tissue or administration of a suspension by aerosolinto the nasal and/or pulmonary areas. The polymeric matrix compositioncan be selected to have both favorable degradation rates and also to beformed of a material which is bioadhesive, to further increase theeffectiveness of transfer when the device is administered to a vascular,pulmonary, or other surface. The matrix composition also can be selectednot to degrade, but rather, to release by diffusion over an extendedperiod of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

In general, the agents of the invention may be delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the peptide, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the platelet reducing agentis contained in a form within a matrix such as those described in U.S.Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 and (b) diffusionalsystems in which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and5,407,686. In addition, pump-based hardware delivery systems can beused, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for prophylactic treatment of subjects at risk of developing aninfection such as a Staphylococcal infection. Long-term release, as usedherein, means that the implant is constructed and arranged to deliverytherapeutic levels of the active ingredient for at least 30 days, andpreferably 60 days. Long-term sustained release implants are well-knownto those of ordinary skill in the art and include some of the releasesystems described above.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES

S. aureus and S. epidermidis are associated with a wide range ofhospital and community acquired infections. The rise of antibioticresistance drives the development of new therapies to treat and preventthese infections. Adhesion of the bacteria to host tissues or toimplanted prosthetic devices is often important for a successfulStaphylococcal infection. One such adhesion molecule expressed on thesurface of Staphylococci in vivo and found to be a target of protectiveantibodies is poly-N acetyl-glucosamine (PNAG). This adhesion moleculeis expressed and employed by other bacteria such as but not limited toE. coli.

Experimental Procedures Hybridomas:

Blood was collected from patients after the onset of S. aureus infectionand peripheral blood mononuclear cells (PBMC) were isolated from theblood samples using Ficoll Hypaque sedimentation. B cells werestimulated by overnight exposure to Epstein-Barr virus (EBV) producedfrom the B95.8 cell line as described by Posner et al. (Posner et al.Epstein Barr virus transformation of peripheral blood B cells secretingantibodies reactive with cell surface antigens. Autoimmunity. 1990;8(2):149-58.) After 24 hours, the cells were washed and dispersed into96 well plates at a concentration of 1×10⁶ PBMC/well in 100 μl of growthmedia (RPMI1640 supplemented with 20% FBS) containing 10% LymphocyteConditioned Medium (LyCM, prepared from human PBMC stimulated for 48hours with phytohemagglutinin). After 5 days, an additional 100 μl ofgrowth media supplemented with 10% LyCM was added. EBV stimulated cellswere then fed weekly by removal of 100 μl of spent media and theaddition of 100 μl of growth media supplemented with 10% LyCM.

When the wells were densely seeded (as evidenced by growth over 80% ofthe bottom of the well surface and the appearance of a pH change in themedia indicative of cellular growth), the cultures were screened forproduction of specific antibody to PNAG/dPNAG by ELISA. The cells fromsingle individual wells giving a positive reaction for antibody werethen dispersed into 48 wells of a tissue culture plate and after severaldays of growth the supernates tested for reactivity with PNAG/dPNAGantigen.

Cultures that continued to test positive by ELISA were then fused withthe human-mouse myeloma cell line HMMA 2.5 to generate hybridomas aspreviously described (Posner et al. The construction and use of ahuman-mouse myeloma analogue suitable for the routine production ofhybridomas secreting human monoclonal antibodies. Hybridoma. 1987December; 6(6):611-25). After fusion, cells were cultured in microwellplates with growth medium (RPMI 1640 supplemented with 20% FBS andhypoxanthine-aminopterin-thymidine (HAT) and oubain) for selection offused cells. These cultures were fed at weekly intervals and screened byELISA for antibody production.

Hybridomas were cloned at a density of 1 cell/well, wells with positivegrowth screened by ELISA for specific antibody, and wells containingpositive antibody-producing hybridomas expanded into wells in tissueculture plates of increasing volume, then flasks of increasing volume toobtain cloned cell lines. Three hybridomas, designated F598, F628 andF630, producing human IgG2 antibodies that bound to either PNAG, dPNAGor both were recovered.

Chemical Modification of PNAG:

To remove the majority of the N- and O-substituents, purified PNAG wasdissolved in 5M NaOH to a final concentration of 0.5 mg/ml and incubatedfor 18 hr at 37° C. with stirring. The strong base solution was thenneutralized with 5N HCl, to a final pH between 6 and 8, and dialyzedagainst dH₂O for 24 hrs. The final product was obtained by freeze dryingthe sample.

ELISAs:

Immulon 4 microtiter plates were coated with 100 μl of the optimalbinding concentration of each antigen (0.6 μg/ml for native PNAG and 3μg/ml for dPNAG) in sensitizing buffer (0.2M NaH₂PO₄, 0.2M Na₂HPO₄,0.02% azide) and incubated overnight at 4° C. Plates were washed 3× withPBS/0.05% tween and blocked with 200 μl of 5% skim milk in PBS, and thenincubated overnight at 4° C. The plates were washed and purified MAbswere added at various concentrations, diluted in 5% skim milk/0.05%tween in PBS (dilution buffer). The plates were then incubated for 1 hrat 37° C. and washed. 100 μl of secondary antibody (anti-human IgG wholemolecule-conjugated to alkaline phosphatase (AP) and obtained from ICN)was added at a 1:1000 dilution made in the dilution buffer. The plateswere incubated at 37° C. for 1 hr and washed. 100 μl of p-NitrophenylPhosphate at a concentration of 1 mg/ml in substrate buffer (800 mgNaHCO₃, 1.46 g Na₂CO₃, 10 mg MgCl, 20 mg Na₃N in 500 ml H₂O) was addedand the plates were incubated at room temperature for 30 min. The plateswere read at 405 nm. Purified human IgG from Sigma was used as astandard to quantify MAbs on plates sensitized with anti-human IgG.

Complement Deposition Assay:

Microtiter plates were prepared as for the ELISA assay. After incubationwith the MAb and washing, normal human sera absorbed with threedifferent S. aureus strains was used as a source of complement at adilution of 1:50 in dilution buffer. The plates were incubated for 15min at 37° C. After washing the plate, goat anti-human C3 antibody wasadded at a concentration of 1:2000 and incubated for one hour at 37° C.An anti goat IgG-AP conjugate was added at 1:2000 and incubated at 37°C. for one hour. The plates were developed essentially as for the ELISAassay, except only for a 15 min duration.

Opsonophagocytic Assays:

Opsonophagocytic killing assays have been described previously. (SeeAmes et al. Infection and Immunity 49:281-285, 1985 and Maira-Litran etal. Infect Immun. 70(8):4433-4440, 2002.) The target strain used is Mn8(S. aureus). The target strain was grown to an optical density at awavelength of 650 nm (OD₆₅₀) of 0.4 and diluted to 1:100 for the assay.Complement (obtained from an infant rabbit via a commercial source suchas Accurate Chemical And Scientific Corp. Westbury, N.Y. 11590, and usedat a 1:15 dilution) was absorbed for 1 hr with the Mn8m strain of S.aureus (resuspended to an OD₆₅₀ of 1.0). Polymorphonuclear cells (PMNs)were separated from freshly drawn human blood using Heparin/dextran (1:1mix). PMNs were used at a concentration of 5×10⁶ cells/ml. Solutionswith the monoclonal antibodies at various concentrations are used as theantibody source. One hundred μl of each component (monoclonal antibodysolution, PMNs, complement, target bacteria) were added together andthen incubated for 1.5 hr at 37° C., while rotating. Supernates weretaken and dilutions made and then aliquots plated on trypic soy agar(TSA), generally using supernate dilutions of 1:100 and 1:1000. Afterincubating overnight at 37° C., bacterial colonies were counted andlevels of killing calculated.

Cloning of Antibody Variable Regions:

RNA extraction from each hybridoma was performed on ˜6×10⁶ cells usingthe RNAeasy kit from Qiagen. 1 μg of total RNA was reverse transcribedinto cDNA using a Qiagen Omniscript kit. 1 μl of cDNA product was usedas a template for the PCR reactions. Each reaction consisted of 50 μl ofInvitrogen Hi fi mix, 100 pmoles of each nucleotide primer and 1 μl ofcDNA template. ˜30 PCR cycles were performed with the followingprotocol: 94° C. for 30 sec, cycle: 94° C. for 30 sec, 65° C. for 30sec, 72° C. for 1 min, final extension 72° C. for 5 min. PCR productswere sequenced and searched using the Ig BLAST program against knowngermline sequences available on the NCBI database.

Primers used to clone antibody variable regions from the hybridoma celllines deposited with the ATCC under Accession No. PTA-5931, PTA-5932 andPTA-5933 on Apr. 21, 2004, are as follows: (5′-3′ with restriction sitesunderlined and starting ATGs in bold):

F598 light chain (SEQ ID NO: 49)lambda constant: GACCGAGGGGGCAGCCTTGGGCTGACCTAGG  (SEQ ID NO: 50)Hu lambda sig 5: AGATCTCTCACCATGGCATGGATCCCTCTCTTC  F598 heavy chain(SEQ ID NO: 51) Heavy chain constant: TGGGCCCTTGGTGCTAGCTGAGGAGAC (SEQ ID NO: 52) VH7LDRHU: GTCGAC ATGAAACATCTGTGGTTCTTC  F628 light chain(SEQ ID NO: 49) lambda constant: GACCGAGGGGGCAGCCTTGGGCTGACCTAGG (SEQ ID NO: 53) Hu lambda sig 1: AGATCTCTCACCATGGCCRGCTTCCCTCTCCTC F628 heavy chain (SEQ ID NO: 51)Heavy chain constant: TGGGCCCTTGGTGCTAGCTGAGGAGAC  (SEQ ID NO: 52)VH7LDRHU: GTCGAC ATGAAACATCTGTGGTTCTTC  F630 light chain (SEQ ID NO: 49)lambda constant: GACCGAGGGGGCAGCCTTGGGCTGACCTAGG  (SEQ ID NO: 50)Hu lambda sig 5: AGATCTCTCACCATGGCATGGATCCCTCTCTTC  F630 heavy chain(SEQ ID NO: 51) Heavy chain constant: TGGGCCCTTGGTGCTAGCTGAGGAGAC (SEQ ID NO: 54) VH1LDRHU: GTCGAC ATGGACTGGACCTGGA 

In Vivo Bacterial Challenge Assays:

Mice were intravenously (IV) administered MAb F598 which binds to bothPNAG and dPNAG, or a control, non-PNAG/dPNAG binding human IgG1 MAb toP. aeruginosa alginate or MEP (designated MAb F429) to induce passiveimmunity. Twenty-four hours later, mice were challenged with S. aureus(5×10⁷ CFU/mouse) by the same route of administration as the MAb.

CFU levels in blood 2 hours after infection were used as the measure ofefficacy of the MAb administered for inducing passive immunity againstS. aureus.

Results MAb Sequences:

The amino acid and nucleotide sequences for the variable regions and CDRof the MAbs F598, F628 and F630 are shown below. CDR regions areunderlined and constant regions are italicized.

Ia. F598 Heavy Chain Variable region Nucleotide and Amino Acid SequenceAlignment

CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCGQ   V   Q   L   Q   E   S   G   P   G  L   V   K   P   SGAG ACC CTG TCC CTC ACC TGC ACT GTT TCT GGT GGC TCC ATC AGTE   T   L   S   L   T   C   T   V   S  G   G   S   I   SGGT TAC TAC TGG AGT TGG ATC CGG CAG CCC CCA GGG AAG GGA CTGG   Y   Y   W   S    W   I   R   Q   P  P   G   K   G   LGAG TGG ATT GGG TAT ATT CAT TAT AGT AGG AGC ACC AAC TCC AACE   W   I   G    Y   I   H   Y   S   R   S   T   N   S   NCCC GCC CTC AAG AGT CGA GTC ACC ATA TCA TCA GAC ACG TCC AAGP   A   L   K   S    R   V   T   I   S   S   D   T   S   KAAC CAG CTC TCC CTG AGA CTG AGC TCA GTG ACC GCT GCG GAC ACGN   Q   L   S   L   R   L   S   S   V   T   A   A   D   TGCC GTG TAT TAC TGT GCG AGA GAT ACC TAT TAC TAT GAT AGT GGTA   V   Y   Y   C   A   R    D   T   Y   Y   Y   D   S   GGAT TAT GAG GAT GCT TTT GAT ATT TGG GGC CAA GGG ACA ATG GTCD   Y   E   D   A   F   D   I    W   G   Q   G   T   M   VACC GTC TCC TCA (SEQ ID NO: 25) T   V   S   S (SEQ ID NO: 1)Ib. F598 Heavy Chain Variable Region Amino Acid Sequence

(SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGGSIS GYYWS WIRQPP GKGLEWIGYIHYSRSTNSNPALKS RVTISSDTSKNQLSLRLS SVTAADTAVYYCAR DTYYYDSGDYEDAFDIWGQGTMVTVSS (SEQ ID NO: 55) QVQLQESGPGLVKPSETLSLTCTVSGGSIS GYYWS WIRQPPGKGLEWIG YIHYSRSTNSNPALKS RVTISSDTSKNQLSLRLSS VTAADTAVYYCARDTYYYDSGDYEDAFDI WGQGTMVTVSSAS Ic. F598 Heavy Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 25)CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCGGAG ACC CTG TCC CTC ACC TGC ACT GTT TCT GGT GGC TCC ATC AGTGGT TAC TAC TGG AGT TGG ATC CGG CAG CCC CCA GGG AAG GGA CTGGAG TGG ATT GGG TAT ATT CAT TAT AGT AGG AGC ACC AAC TCC AACCCC GCC CTC AAG AGT CGA GTC ACC ATA TCA TCA GAC ACG TCC AAGAAC CAG CTC TCC CTG AGA CTG AGC TCA GTG ACC GCT GCG GAC ACGGCC GTG TAT TAC TGT GCG AGA GAT ACC TAT TAC TAT GAT AGT GGTGAT TAT GAG GAT GCT TTT GAT ATT TGG GGC CAA GGG ACA ATG GTCACC GTC TCC TCA (SEQ ID NO: 56)CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG CCT TCGGAG ACC CTG TCC CTC ACC TGC ACT GTT TCT GGT GGC TCC ATC AGTGGT TAC TAC TGG AGT TGG ATC CGG CAG CCC CCA GGG AAG GGA CTGGAG TGG ATT GGG TAT ATT CAT TAT AGT AGG AGC ACC AAC TCC AACCCC GCC CTC AAG AGT CGA GTC ACC ATA TCA TCA GAC ACG TCC AAGAAC CAG CTC TCC CTG AGA CTG AGC TCA GTG ACC GCT GCG GAC ACGGCC GTG TAT TAC TGT GCG AGA GAT ACC TAT TAC TAT GAT AGT GGTGAT TAT GAG GAT GCT TTT GAT ATT TGG GGC CAA GGG ACA ATG GTCACC GTC TCC TCA GCT AGCIIa. F598 Light Chain Variable Region Amino Acid and Nucleotide SequenceAlignment

CAG CTT GTG CTG ACT CAG TCG CCC TCT GCC TCT GCCQ   L   V   L   T   Q   S   P   S   A   S   ATCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGS   L   G   A   S   V   K   L   T   C    T   LAGC AGT GGC CAC AGC AAC TAC GCC ATC GCT TGG CATS   S   G   H   S   N   Y   A   I   A    W   HCAG CAG CAG CCA GGG AAG GGC CCT CGC TAC TTG ATGQ   Q   Q   P   G   K   G   P   R   Y   L   MAAG GTT AAC AGA GAT GGC AGC CAC ATC AGG GGG GAC K   V   N   R   D   G   S   H   I   R   G   DGGG ATC CCT GAT CGC TTC TCA GGC TCC ACC TCT GGGG   I   P   D   R   F   S   G   S   T   S   GGCT GAG CGT TAC CTC ACC ATC TCC AGT CTC CAG TCTA   E   R   Y   L   T   I   S   S   L   Q   SGAA GAT GAG GCT GAC TAT TAC TGT CAG ACC TGG GGCE   D   E   A   D   Y   Y   C    Q   T   W   GGCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG A   G   I   R   V   F   G   G   G   T   K   L ACC GTC CTA GGT (SEQ ID NO: 26)T   V   L   G   (SEQ ID NO: 2)IIb. F598 Light Chain Variable Region Amino Acid Sequence

(SEQ ID NO: 2) QLVLTQSPSASASLGASVKLTC TLSSGHSNYAIA WHQQQPGKGPRYL MKVNRDGSHIRGD GIPDRFSGSTSGAERYLTISSLQSEDEA DYYC QT WGAGIRV FGGGTKLTVLG(SEQ ID NO: 57) QLVLTQSPSASASLGASVKLTC TLSSGHSNYAIA WHQQQPGKGPRYL MKVNRDGSHIRGD GIPDRFSGSTSGAERYLTISSLQSEDEA DYYC QT WGAGIRVFGGGTKLTVLGQPKAAPSVIIc. F598 Light Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 26) CAG CTT GTG CTG ACT CAG TCG CCC TCT GCC TCT GCCTCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGAGC AGT GGC CAC AGC AAC TAC GCC ATC GCT TGG CATCAG CAG CAG CCA GGG AAG GGC CCT CGC TAC TTG ATGAAG GTT AAC AGA GAT GGC AGC CAC ATC AGG GGG GACGGG ATC CCT GAT CGC TTC TCA GGC TCC ACC TCT GGGGCT GAG CGT TAC CTC ACC ATC TCC AGT CTC CAG TCTGAA GAT GAG GCT GAC TAT TAC TGT CAG ACC TGG GGCGCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGTIIIa. F628 Heavy Chain Variable Region Amino Acid and NucleotideSequence Alignment

CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTGQ   V   Q   L   Q   E   S   G   P   G   L   VAAG CCT TCG GAG ACC CTG TCC CTC ACG TGC ACT GTCK   P   S   E   T   L   S   L   T   C   T   VTCT GGT GGC TCC ATC AGT AAT TAC TAC TGG AGT TGG S   G   G   S   I   S   N   Y   Y   W   S    W ATC CGG CAG TCC CCA GGG AGG GGA CTG GAG TGG ATTI   R   Q   S   P   G   R   G   L   E   W   IGGG TAT ATC CAT TAT AGT GGG AGC ACC AAC TCC AAT G   Y   I   H   Y   S   G   S   T   N   S   NCCA TCC CTC AAG AGT CGA GTC ACC ATA TCA GTT GAC P   S   L   K   S   R   V   T   I   S   V   DACG TCC AAG AAC CAG GTC TCC CTG AAG CTG GGC TCTT   S   K   N   Q   V   S   L   K   L   G   SGTG ACC GCT GCG GAC ACG GCC ATA TAT TAC TGT GCGV   T   A   A   D   T   A   I   Y   Y   C   AAGA GAT ACT TAC TAT GAA AGT AGT GGT CAT TGG TTC R   D   T   Y   Y   E   S   S   G   H   W   FGAC GGT TTG GAC GTC TGG GGC CAA GGG ACC TCG GTC D   G   L   D   V   W   G   Q   G   T   S   V ACC GTC TCC TCA (SEQ ID NO: 27)T   V   S   S   (SEQ ID NO: 3)IIIb. F628 Heavy Chain Variable Region Amino Acid Sequence

(SEQ ID NO: 3) QVQLQESGPGLVKPSETLSLTCTVSGGSIS NYYWS WIRQSPGRGLEWI GYIHYSGSTNSNPSLKS RVTISVDTSKNQVSLKLGSVTAADTAIYYCA R DTYYESSGHWFDGLDVWGQGTSVTVSS (SEQ ID NO: 58) QVQLQESGPGLVKPSETLSLTCTVSGGSIS NYYWSWIRQSPGRGLEWI G YIHYSGSTNSNPSLKS RVTISVDTSKNQVSLKLGSVTAADTAIYYCA RDTYYESSGHWFDGLDV WGQGTSVTVSSASTKGPIIIc. F628 Heavy Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 27) CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTGAAG CCT TCG GAG ACC CTG TCC CTC ACG TGC ACT GTCTCT GGT GGC TCC ATC AGT AAT TAC TAC TGG AGT TGGATC CGG CAG TCC CCA GGG AGG GGA CTG GAG TGG ATTGGG TAT ATC CAT TAT AGT GGG AGC ACC AAC TCC AATCCA TCC CTC AAG AGT CGA GTC ACC ATA TCA GTT GACACG TCC AAG AAC CAG GTC TCC CTG AAG CTG GGC TCTGTG ACC GCT GCG GAC ACG GCC ATA TAT TAC TGT GCGAGA GAT ACT TAC TAT GAA AGT AGT GGT CAT TGG TTCGAC GGT TTG GAC GTC TGG GGC CAA GGG ACC TCG GTC ACC GTC TCC TCA(SEQ ID NO: 59) CAG GTG CAG CTG CAG GAG TCG GGC CCA GGA CTG GTGAAG CCT TCG GAG ACC CTG TCC CTC ACG TGC ACT GTCTCT GGT GGC TCC ATC AGT AAT TAC TAC TGG AGT TGGATC CGG CAG TCC CCA GGG AGG GGA CTG GAG TGG ATTGGG TAT ATC CAT TAT AGT GGG AGC ACC AAC TCC AATCCA TCC CTC AAG AGT CGA GTC ACC ATA TCA GTT GACACG TCC AAG AAC CAG GTC TCC CTG AAG CTG GGC TCTGTG ACC GCT GCG GAC ACG GCC ATA TAT TAC TGT GCGAGA GAT ACT TAC TAT GAA AGT AGT GGT CAT TGG TTCGAC GGT TTG GAC GTC TGG GGC CAA GGG ACC TCG GTCACC GTC TCC TCA GCT AGC ACCIVa. F628 Light Chain Variable Region Amino Acid and Nucleotide SequenceAlignment

CAG CCT GTG CTG ACT CAG TCG CCC TCT GCC TCT GCCQ   P   V   L   T   Q   S   P   S   A   S   ATCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGS   L   G   A   S   V   K   L   T   C    T   LGAC AGT GAA CAC AGC AGA TAC ACC ATC GCA TGG CATD   S   E   H   S   R   Y   T   I   A    W   HCAG CAG CAG CCA GAG AAG GGC CCT CGG TAC CTG ATGQ   Q   Q   P   E   K   G   P   R   Y   L   MAAG GTT AAG AGT GAT GGC AGT CAC AGC AAG GGG GAC K   V   K   S   D   G   S   H   S   K   G   DGGC ATT ACT GAT CGC TTC TCA GGC TCC AGC TCT GGGG   I   T   D   R   F   S   G   S   S   S   GGCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCTA   E   R   Y   L   T   I   S   S   L   Q   SGAG GAT GAG GCT GAC TAT TAC TGT CAG ACT TGG GGCE   D   E   A   D   Y   Y   C    Q   T   W   GCCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG P   G   I   R   V   F   G   G   G   T   K   L ACC GTC CTA (SEQ ID NO: 28)T   V   L   (SEQ ID NO: 4)IVb. F628 Light Chain Variable Region Amino Acid Sequence

(SEQ ID NO: 4) QPVLTQSPSASASLGASVKLTC TLDSEHSRYTIA WHQQQPEKGPRYLM KVKSDGSHSKGD GITDRFSGSSSGAERYLTISSLQSEDEA DYYC QTW GPGIRV FGGGTKLTVLIVc. F628 Light Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 28) CAG CCT GTG CTG ACT CAG TCG CCC TCT GCC TCT GCCTCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGGAC AGT GAA CAC AGC AGA TAC ACC ATC GCA TGG CATCAG CAG CAG CCA GAG AAG GGC CCT CGG TAC CTG ATGAAG GTT AAG AGT GAT GGC AGT CAC AGC AAG GGG GACGGC ATT ACT GAT CGC TTC TCA GGC TCC AGC TCT GGGGCT GAG CGC TAC CTC ACC ATC TCC AGC CTC CAG TCTGAG GAT GAG GCT GAC TAT TAC TGT CAG ACT TGG GGCCCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG ACC GTC CTAVa. F630 Heavy Chain Variable Region Amino Acid and Nucleotide SequenceAlignment

CAG GTT CAG CTG GTG CAG TCT GGA GCT GAG ATG AAGQ   V   Q   L   V   Q   S   G   A   E   M   KAGG CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GCTR   P   G   A   S   V   K   V   S   C   K   ATCT GGT TAC ACC TTT ACC AAC TTT GGT ATC AGT TGG S   G   Y   T   F   T   N   F   G   I   S    W GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATAV   R   Q   A   P   G   Q   G   L   E   W   IGGA TGG GTC AGC ACT TAC AAT GGT CGC ACA AAT TAT G   W   V   S   T   Y   N   G   R   T   N   YGCA CAG AAG TTC CGG GGC AGA GTC ACC ATG ACC ACA A   Q   K   F   R   G   R   V   T   M   T   T GAC ACA TCC ACG AAC ACA GCG TAC ATG GAA CTG AGGD   T   S   T   N   T   A   Y   M   E   L   RAGC CTG GGA TCT GAC GAC ACG GCC GTC TTT TAC TGTS   L   G   S   D   D   T   A   V   F   Y   CGCG AGA GAT TAC TAT GAG ACT AGT GGT TAC GCC TAT A   R   D   Y   Y   E   T   S   G   Y   A   YGAT GAT TTT GCG ATC TGG GGC CAA GGG ACA TTG GTC D   D   F   A   I   W   G   Q   G   T   L   V ACC GTC TCC TCA (SEQ ID NO: 29)T   V   S   S   (SEQ ID NO: 5)Vb. F630 Heavy Chain Variable Region Amino Acid Sequence

(SEQ ID NO: 5) QVQLVQSGAEMKRPGASVKVSCKASGYTFT NFGIS WVRQAPGQGLEWI GWVSTYNGRTNYAQKFRG RVTMTTDTSTNTAYMELRSLGSDDTAVFYC AR DYYETSGYAYDDFAIWGQGTLVTVSSVc. F630 Heavy Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 29) CAG GTT CAG CTG GTG CAG TCT GGA GCT GAG ATG AAGAGG CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GCTTCT GGT TAC ACC TTT ACC AAC TTT GGT ATC AGT TGGGTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATAGGA TGG GTC AGC ACT TAC AAT GGT CGC ACA AAT TATGCA CAG AAG TTC CGG GGC AGA GTC ACC ATG ACC ACAGAC ACA TCC ACG AAC ACA GCG TAC ATG GAA CTG AGGAGC CTG GGA TCT GAC GAC ACG GCC GTC TTT TAC TGTGCG AGA GAT TAC TAT GAG ACT AGT GGT TAC GCC TATGAT GAT TTT GCG ATC TGG GGC CAA GGG ACA TTG GTC ACC GTC TCC TCAVIa. F630 Light Chain Variable Region Amino Acid and Nucleotide SequenceAlignment

CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT GCTQ   L   V   L   T   Q   S   P   S   A   S   ATCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGS   L   G   A   S   V   K   L   T   C    T   LAGC AGT GGG CAC AGC ACC TAC GCC ATC GCG TGG CATS   S   G   H   S   T   Y   A   I   A    W   HCAG CAG CAG CCA CTG AGG GGT CCT CGT TTC TTG ATGQ   Q   Q   P   L   R   G   P   R   F   L   MAAA GTC AAC AGT GAT GGC AGC CAC ACC AAG GGG GAC K   V   N   S   D   G   S   H   T   K   G   DGGG ATC CCT GAT CGC TTC TCA GGC TCC AGT TCT GGGG   I   P   D   R   F   S   G   S   S   S   GGCT GAG CGC TAC CTC TCC ATC TCC AGC CTC CAG TCTA   E   R   Y   L   S   I   S   S   L   Q   SGAA GAT GAG TCT GAC TAT TAC TGT CAG ACG TGG GGCE   D   E   S   D   Y   Y   C    Q   T   W   GCCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG P   G   I   R   V   F   G   G   G   T   K   L ACC GTC CTA GGT (SEQ ID NO: 30)T   V   L   G   (SEQ ID NO: 6)VIb. F630 Light Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 6) QLVLTQSPSASASLGASVKLTC TLSSGHSTYAIA WHQQQPLRGPRFLM KVNSDGSHTKGD GIPDRFSGSSSGAERYLSISSLQSEDESDYYC QTWG PGIRV FGGGTKLTVLG(SEQ ID NO: 60) QLVLTQSPSASASLGASVKLTC TLSSGHSTYAIA WHQQQPLRGPRFLM KVNSDGSHTKGD GIPDRFSGSSSGAERYLSISSLQSEDESDYYC QTWG PGIRVFGGGTKLTVLGQPKAAPSVVIc. F630 Light Chain Variable Region Nucleotide Sequence

(SEQ ID NO: 30) CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT GCTTCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGAGC AGT GGG CAC AGC ACC TAC GCC ATC GCG TGG CATCAG CAG CAG CCA CTG AGG GGT CCT CGT TTC TTG ATGAAA GTC AAC AGT GAT GGC AGC CAC ACC AAG GGG GACGGG ATC CCT GAT CGC TTC TCA GGC TCC AGT TCT GGGGCT GAG CGC TAC CTC TCC ATC TCC AGC CTC CAG TCTGAA GAT GAG TCT GAC TAT TAC TGT CAG ACG TGG GGCCCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGT(SEQ ID NO: 61) CAG CTT GTG CTG ACT CAA TCG CCC TCT GCC TCT GCTTCC CTG GGA GCC TCG GTC AAG CTC ACC TGC ACT CTGAGC AGT GGG CAC AGC ACC TAC GCC ATC GCG TGG CATCAG CAG CAG CCA CTG AGG GGT CCT CGT TTC TTG ATGAAA GTC AAC AGT GAT GGC AGC CAC ACC AAG GGG GACGGG ATC CCT GAT CGC TTC TCA GGC TCC AGT TCT GGGGCT GAG CGC TAC CTC TCC ATC TCC AGC CTC CAG TCTGAA GAT GAG TCT GAC TAT TAC TGT CAG ACG TGG GGCCCT GGC ATT CGA GTG TTC GGC GGA GGG ACC AAG CTGACC GTC CTA GGT CAG CCC AAG GCT GCC CCA TCG GTC ACC TGT TCC CGC CTC

Characterization of IgG2 MAbs:

The hybridomas were named from their corresponding fusion numbers: F598,F628, and F630. The antibodies produced from these hybridomas were allIgG2 and lambda types. After purification of the antibodies usingprotein G columns, ELISAs were used to determine differences in epitopespecificities of the MAbs. Chemical modification of native PNAG wasperformed in order to remove certain substituents. Strong base treatment(5M NaOH) results in removal of most of the N-acetyl groups. As seen inFIG. 1 all of the MAbs bind well to the native form of PNAG, althoughwith different binding curves. When PNAG is treated with 5M NaOH toyield dPNAG, F598 MAb binds with the greatest activity (FIG. 2). Thisresult suggests that F598 has specificity for the backbone epitopes ofPNAG and does not require N- and O-acetylated groups to bind to the PNAGpolymer. MAbs F630 and F628 bind poorly to dPNAG suggesting that theirspecificities require the acetates found in the native form of PNAG.

Competition ELISAs were used to determine the relative bindingactivities of the MAbs. FIG. 3 shows that the relative binding activityof the MAbs are:

F598>F628>F630.

Creation and Characterization of IgG1 Switched MAbs:

Human IgG1 isotype antibodies can fix complement onto the surface of anantigen better than antibodies of the IgG2 isotype. Therefore, cloningof the MAb variable regions and production of the IgG1 isotype wasperformed. Primers directed at the IgG2 constant region and primersspecific for the 5′ end of the variable regions identified in theoriginal hybridomas (see listing of primers herein under “Cloning ofantibody variable regions”) were used to obtain PCR products from cDNApreparations made from mRNA isolated from the original IgG2 hybridomacell lines. PCR products were sequenced and analyzed to determine thegermline genes that most likely gave rise to each antibody. As shown inTable 1, there are common germline genes that are used by the hybridomasin making antibodies directed to PNAG and/or dPNAG. As shown, MAbs F598,F628 and F630 use the same light chain germline genes and heavy chain Dregions. The only difference is the V gene used to produce the heavychain of MAb F630 and the J gene used to produce the heavy chain of MAbF628; the remainder of the germline genes are identical for the MAbs.

TABLE 1 Hybridoma H or L chain Germline Genes F598L IGLV4-69 or V5-6IGLJ3 or IGLJ2 F598H IGHV4-59 IGHD3-22 IGHJ3 F628L IGLV4-69 or V5-6IGLJ3 or IGLJ2 F628H IGHV4-59 IGHD3-22 IGHJ6 F630L IGLV4-69 or V5-6IGLJ3 or IGLJ2 F630H IGHV1-18 IGHD3-22 IGHJ3

The DNA encoding the entire variable regions encompassing V, J and Dsegments for the heavy chain and V and J segments for the light chain ofeach of the MAbs (F598, F728 and F630) was cloned into the TCAE6 vector,which contains the kappa light chain and IgG1 heavy chain human constantregions. The initial constructs maintained the original pairing of theheavy and light chain genes obtained from the original hybridomas.Plasmid DNA containing each of the constructs were transfected into CHOcells and the resulting IgG1 MAbs were purified and characterized. Asseen in FIGS. 4 and 5, all of the IgG1 MAbs have identical bindingcurves to PNAG when compared to the original IgG2 MAbs, however the IgG1constructs of MAbs F628 and F630 have lost some of their ability to bindto dPNAG (e.g., compare with FIGS. 1 and 2).

To test whether the IgG1 MAbs have more functional complement activatingactivity than the IgG2 parental MAbs, complement deposition assays wereperformed. The complement deposition assay is essentially an ELISA assaythat measures the deposition of complement protein C3 when human serumis added to the reaction mixture. As shown in FIG. 6, all of the IgG1MAbs have better complement fixing activity than the parental IgG2 MAbs.The extent of the increase in complement fixation depends on the MAb.For MAb F598, which has the highest binding activity to PNAG and dPNAG,there is only a slight increase in activity of the IgG1 over the IgG2isotype. For MAbs F628 and F630, the IgG1 MAbs have at least double thecomplement deposition activity than the parental IgG2 MAbs.

Opsonophagocytic Activity:

The opsonophagocytic activity of monoclonal antibodies F598, F628 andF630 in both the IgG1 and IgG2 forms (614 of MAb) was tested against S.aureus strain Mn8. Monoclonal antibody F598 showed the highest level ofreduction (i.e., killing) in CFU when the IgG1 form was used (FIG. 7).

Passive Protection Against Infection:

Administration of the MAb F598 that binds to both PNAG and dPNAG to mice24 hours prior to challenge with S. aureus strain Mn8 resulted in a 68%reduction 2 hours following infection in the number of CFU/ml blood ascompared to mice receiving a MAb to an irrelevant antigen, P. aeruginosaalginate (significance of P=0.002) (FIG. 8A). FIG. 8B shows the CFU ofS. aureus per ml of blood for each individual animal given either thecontrol MAb or the MAb F598g1. Administration of 800 μg MAb F598 per FVBmouse 4 hours prior to intraperitoneal (IP) challenge with 5×10⁸ CFU S.aureus (Mn8 strain) resulted in increased survival compared to miceadministered a control MAb (F429 specific to P. aeruginosa). FIG. 8Cshows the results of these experiments. At five days after bacterialchallenge, all mice that received F598 and only about 20% of micereceiving control MAb were alive (8 mice per group).

E. coli Urinary Tract Infection Isolates:

Eighteen E. coli clinical urinary tract infection (UTI) isolates wereisolated and tested for the presence of the pga locus by PCR and PNAGexpression by immunoblot using antisera raised to S. aureus PNAG. Theclinical isolates were grown in culture and either DNA was extracted bystandard techniques for use in PCR or cells were subjected to EDTAextraction (boiling for 5 minutes) once cells were in stationary phase.Seventeen of the eighteen isolates carried pga genes as determined byPCR. Based on the immunoblot results, of these seventeen, about onethird were characterized as expressing relatively high levels of PNAG,about one third were characterized as expressing relatively intermediate(or moderate) levels of PNAG and the remaining one third werecharacterized as expressing relatively low levels of PNAG. In addition,over-expression of the pga locus resulted in enhanced production ofPNAG. FIG. 9 shows the results of this immunoblot. Strain “H” expressesundetectable levels of PNAG and does not have a pga locus. The slot atthe upper right hand corner represents the pga over-expressing strain ofE. coli.

FIG. 10 shows the level of opsonic killing of the afore-mentioned E.coli clinical UTI isolates using a polyclonal antiserum raised againstS. aureus dPNAG. BW represents a wild type E. coli strain, pgarepresents an E. coli strain with the pga locus deleted, and pga⁺⁺represents a pga over-expressing strain of E. coli. The level of killingroughly correlates with the level of PNAG expression by the E. coliisolate.

FIGS. 11A and 11B show the level of opsonic killing of a high PNAGexpressing E. coli strain (strain U) and an intermediate PNAG expressingE. coli strain (strain P) by polyclonal antiserum raised against dPNAGand PNAG. At all antiserum dilutions tested, the anti-dPNAG was moreeffective at killing either strain than the anti-PNAG antiserum.

FIG. 12 shows the opsonophagocytic activity of MAb F598 against variousStaphylococcal strains and an E. coli strain by MAb F598, F628 and F630(6 μg/ml of MAb per assay).

Ica Locus Mutation:

FIGS. 13 and 14 shows the results of killing by MAbs F598 and F628 of S.aureus strains having mutant ica loci. S. aureus strain 10833 wasdeleted for the ica locus (10833Δica) then transformed with a plasmidcarrying wild-type ica isolated from S. aureus Mn8m (pMuc,PNAG-over-producer) or with pMuc with the icaB gene deleted (pMucΔicaB),as shown in FIG. 13. S. aureus strain 10833 (wild type) and 10833(picaB) are shown in FIG. 14. Strain 10833 (picaB) over-expresses theicaB gene from a plasmid using the constitutive promoter from the icalocus of S. aureus Mn8m (PNAG-over-producer). The icaB gene is theenzyme believed responsible for deacetylating PNAG. Deletion of the icaBgene affects killing by MAb F598 but not MAb F628. In the absence of theicaB gene, killing by MAb F598 is reduced (FIG. 13). Over-expression ofthe icaB gene results in enhanced killing by MAb F598 with little or noeffect on MAb 628 killing.

Conclusions

ELISAs using chemically modified PNAG highlighted differences in thespecificity of three fully human MAbs directed at the native form ofPNAG. MAb F598 was found to recognize PNAG and dPNAG and so is specificfor the backbone of the molecule. MAbs F628 and F630 apparentlyrecognize acetate-specific epitopes. Competition ELISAs reveal that therelative binding activities of the MAbs ranks F598 with the highestbinding activity followed by F628 and then F630. Cloning of the variableregions reveals that there is gene restriction usage for producingantibodies to PNAG and/or dPNAG. Changing the constant region of theMAbs from gamma 2 to gamma 1 resulted in identical binding to PNAG, butreduced the ability of 2 of the 3 MAbs to bind to dPNAG. Finallychanging the constant region to gamma 1 resulted in an increased abilityof the MAbs to fix complement, however this increase was most dramaticfor MAbs F628 and F630 which have lower binding activity. Evaluation ofthe protective efficacy of MAb F598g1 showed administration this productto mice 24 hours before IV challenge with live S. aureus strain Mn8resulted in a 68% reduction in the levels of Staphylococci in the blood2 hours after infection. Administration of MAb F598 to mice 4 hoursbefore IP challenge with live S. aureus strain Mn8 resulted in increasedsurvival as compared to control MAb treated mice.

Equivalents

The foregoing written specification is to be considered to be sufficientto enable one skilled in the art to practice the invention. Theparticular antibodies and peptides disclosed herein are not to beconstrued as limiting of the invention as they are intended merely asillustrative of particular embodiments of the invention as enabledherein. Therefore, any peptides, antibodies, and antibody fragments thatare functionally equivalent to those described herein are within thespirit and scope of the claims appended hereto. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A composition, comprising an isolated peptide that selectively bindsto Staphylococcal poly-N-acetyl glucosamine (PNAG/dPNAG) and comprisesan amino acid sequence of a Staphylococcal PNAG/dPNAG-binding CDRselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12 or a functionallyequivalent variant thereof, wherein the variant comprises an amino acidsequence that is at least 85% identical to the amino acid sequence ofthe Staphylococcal PNAG/dPNAG-binding CDR.
 2. The composition of claim1, wherein the Staphylococcal PNAG/dPNAG-binding CDR is a StaphylococcalPNAG/dPNAG-binding CDR3 and comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 9 and SEQ ID: NO:
 12. 3. Thecomposition of claim 2, wherein the Staphylococcal PNAG/dPNAG-bindingCDR3 comprises an amino acid sequence of a heavy chain CDR3 according toSEQ ID NO:
 9. 4. The composition of claim 2, wherein the StaphylococcalPNAG/dPNAG-binding CDR3 comprises a polypeptide containing a heavy chainCDR3 of a deposited hybridoma having Accession No. PTA-5931.
 5. Thecomposition of claim 2, wherein the Staphylococcal PNAG/dPNAG-bindingCDR3 comprises an amino acid sequence of a light chain CDR3 according toSEQ ID NO:12.
 6. The composition of claim 2, wherein the StaphylococcalPNAG/dPNAG-binding CDR3 comprises a polypeptide containing a light chainCDR3 of a deposited hybridoma having Accession No. PTA-5931.
 7. Thecomposition of claim 1, wherein the Staphylococcal PNAG/dPNAG-bindingCDR is a Staphylococcal PNAG/dPNAG-binding CDR2 and comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:8 and SEQID NO:11.
 8. The composition of claim 7, wherein the amino acid sequenceof the CDR2 is derived from a deposited hybridoma having Accession No.PTA-5931.
 9. The composition of claim 1, wherein the StaphylococcalPNAG/dPNAG-binding CDR is a Staphylococcal PNAG/dPNAG-binding CDR1 andcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:7 and SEQ ID NO:10.
 10. The composition of claim 9, whereinthe amino acid sequence of a CDR1 is derived from a deposited hybridomahaving Accession No. PTA-5931.
 11. The composition of claim 1, whereinthe isolated peptide comprises an amino acid sequence of SEQ ID NO:1.12. The composition of claim 1, wherein the isolated peptide comprisesan amino acid sequence of a heavy chain variable region derived from adeposited hybridoma having Accession No. PTA-5931.
 13. The compositionof claim 1, wherein the isolated peptide comprises an amino acidsequence of SEQ ID NO:2.
 14. The composition of claim 1, wherein theisolated peptide comprises an amino acid sequence of a light chainvariable region derived from a deposited hybridoma having Accession No.PTA-5931.
 15. The composition of claim 1, wherein the isolated peptideis an isolated antibody or antibody fragment.
 16. The composition ofclaim 15, wherein the isolated antibody or antibody fragment is derivedfrom a deposited hybridoma having Accession No. PTA-5931.
 17. Thecomposition of claim 15, wherein the isolated antibody or antibodyfragment is an intact soluble monoclonal antibody.
 18. The compositionof claim 15, wherein the isolated antibody or antibody fragment is anisolated antibody fragment selected from the group consisting of anF(ab′)₂ fragment, an Fd fragment and an Fab fragment.
 19. Thecomposition of claim 15, wherein the isolated antibody or antibodyfragment enhances opsonophagocytosis of PNAG-expressing bacterialstrains.
 20. The composition of claim 15, wherein the isolated antibodyor antibody fragment enhances opsonophagocytosis of PNAG-expressingStaphylococci.
 21. The composition of claim 20, wherein thePNAG-expressing Staphylococci are S. aureus or S. epidermidis.
 22. Thecomposition of claim 19, wherein the PNAG-expressing bacterial strainsare PNAG-expressing E. coli, Yersinia pestis (Y. pestis), Y.entercolitica, Xanthomonas axonopodis (X. axonopodis), Pseudomonasfluorescens (P. fluorescens), Actinobacillus actinomycetemcomitans (A.actinomycetemcomitans), A. pleuropneumoniae, Ralstonia solanacearum (R.solanacearum), Bordetella pertussis (B. pertussis), B. parapertussis orB. bronchiseptica.
 23. The composition of claim 15, wherein the isolatedantibody or antibody fragment comprises an amino acid sequencecomprising a heavy chain variable region derived from a depositedhybridoma having Accession No. PTA-5931; and an amino acid sequencecomprising a light chain variable region derived from a depositedhybridoma having Accession No. PTA-5931.
 24. The composition of claim15, wherein the isolated antibody or antibody fragment comprises anamino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ IDNO:
 2. 25. The composition of claim 1, wherein the isolated peptide isconjugated to a detectable label.
 26. The composition of claim 25,wherein the detectable label is an in vivo detectable label.
 27. Thecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier.
 28. The composition of claim 27, wherein the isolated peptideis present in an effective amount for inhibiting an infection bybacterial strains expressing PNAG.
 29. The composition of claim 28,wherein the bacterial strains expressing PNAG are selected from thegroup consisting of E. coli, Yersinia pestis (Y. pestis), Y.entercolitica, Xanthomonas axonopodis (X. axonopodis), Pseudomonasfluorescens (P. fluorescens), Actinobacillus actinomycetemcomitans (A.actinomycetemcomitans), A. pleuropneumoniae, Ralstonia solanacearum (R.solanacearum), Bordetella pertussis (B. pertussis), B. parapertussis andB. bronchiseptica.
 30. The composition of claim 27, wherein the isolatedpeptide is present in an effective amount for inhibiting aStaphylococcal infection.
 31. The composition of claim 27, wherein theisolated peptide is present in an effective amount for detectingbacterial strains expressing PNAG in a sample in or from a subject. 32.The composition of claim 31, wherein the bacterial strains expressingPNAG are selected from the group consisting of E. coli, Yersinia pestis(Y. pestis), Y. entercolitica, Xanthomonas axonopodis (X. axonopodis),Pseudomonas fluorescens (P. fluorescens), Actinobacillusactinomycetemcomitans (A. actinomycetemcomitans), A. pleuropneumoniae,Ralstonia solanacearum (R. solanacearum), Bordetella pertussis (B.pertussis), B. parapertussis and B. bronchiseptica.
 33. The compositionof claim 27, wherein the isolated peptide is present in an effectiveamount for detecting Staphylococci in a sample in or from a subject. 34.The composition of claim 33, wherein Staphylococci are S. aureus or S.epidermidis.
 35. The composition of claim 1, wherein the isolatedpeptide selectively binds to Staphylococcal PNAG.
 36. The composition ofclaim 1, wherein the isolated peptide selectively binds toStaphylococcal dPNAG.
 37. An isolated anti-Staphylococcal PNAG/dPNAGmonoclonal antibody produced by an isolated cell producing ananti-Staphylococcal PNAG/dPNAG monoclonal antibody and having ATCCAccession No. PTA-5931, or a fragment thereof.
 38. The isolatedanti-Staphylococcal PNAG/dPNAG monoclonal antibody or the fragmentthereof of claim 37, wherein the fragment is selected from the groupconsisting of an F(ab′)₂ fragment, an Fd fragment, and an Fab fragment.39. The isolated anti-Staphylococcal PNAG/dPNAG monoclonal antibody orthe fragment thereof of claim 37, wherein the fragment enhancesopsonophagocytosis of a bacterial strain expressing PNAG.
 40. Theisolated anti-Staphylococcal PNAG/dPNAG monoclonal antibody or thefragment thereof of claim 37, wherein the fragment enhancesopsonophagocytosis of PNAG-expressing Staphylococci.
 41. The isolatedanti-Staphylococcal PNAG/dPNAG monoclonal antibody or the fragmentthereof of claim 40, wherein the PNAG-expressing Staphylococci is S.aureus or S. epidermidis.
 42. The isolated anti-StaphylococcalPNAG/dPNAG monoclonal antibody or the fragment thereof of claim 39,wherein the PNAG-expressing bacteria is E. coli, Yersinia pestis (Y.pestis), Y. entercolitica, Xanthomonas axonopodis (X. axonopodis),Pseudomonas fluorescens (P. fluorescens), Actinobacillusactinomycetemcomitans (A. actinomycetemcomitans), A. pleuropneumoniae,Bordetella pertussis (B. pertussis), B. parapertussis or B.bronchiseptica.